TLS (Translocated-in-Liposarcoma) Is a High-Affinity Interactor for Steroid, Thyroid Hormone, and Retinoid Receptors

C. Andrew Powers1, Mukul Mathur, Bruce M. Raaka, David Ron and Herbert H. Samuels

Division of Molecular Endocrinology (C.A.P., M.M., B.M.R., H.H.S.) Departments of Medicine (D.R., M.M., B.M.R., H.H.S.), Cell Biology (D.R.), and Pharmacology (M.M., B.M.R., H.H.S.) Skirball Institute of Biomolecular Medicine (D.R.) New York University Medical Center New York, New York 10016


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription complex. In an effort to identify potential coregulators, receptor fusions with glutathione-S-transferase were used to isolate proteins in nuclear extracts capable of binding nuclear hormone receptors. Glutathione-S-transferase fusions with mouse retinoid X receptor-{alpha} enabled the selective isolation of a 65-kDa protein (p65) from nuclear extracts of rat and human cells. Binding of p65 to mouse retinoid X receptor-{alpha} was centered around the DNA-binding domain. p65 also bound regions encompassing the DNA-binding domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65 was identified as TLS (translocated-in-liposarcoma), a recently identified member of the RNP family of nuclear RNA-binding proteins whose members are thought to function in RNA processing. The N-terminal half of TLS bound to thyroid hormone receptor with high affinity while the receptor was bound to appropriate DNA target sites. Functional studies indicated that the N-terminal half of TLS can interact with thyroid hormone receptor in vivo. TLS was originally discovered as part of a fusion protein arising from a chromosomal translocation causing human myxoid liposarcomas. TLS contains a potent transactivation domain whose translocation-induced fusion with a DNA-binding protein (CHOP) yields a powerful transforming oncogene and transcription factor. The transactivation and RNA-binding properties of TLS and the nature of its interaction with nuclear receptors suggest a novel role in nuclear receptor function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors for steroid hormones, 1,25-dihydroxyvitamin D3, T3, and retinoids mediate ligand-dependent transcriptional regulation at target genes containing DNA sequences enabling receptor binding [hormone response elements (HREs)]. These receptors possess a modular structure with six domains (A–F) variously involved in DNA binding, receptor dimerization, ligand binding, and transcriptional regulation (Fig. 1AGo). Ligand binding is dependent on a complex region (domains D, E, and F) that also mediates transcriptional regulation and receptor dimerization (1, 2, 3, 4, 5, 6). The ligand-binding domain (LBD) exhibits considerable variability, as befits the structural diversity of receptor ligands, and contains one of two transactivation regions (AF-2) of nuclear receptors. AF-2 function is strictly ligand dependent and does not require other receptor domains (6, 7, 8). Thus, receptor LBDs fused to the DNA-binding domains (DBDs) of unrelated transcription factors can elicit ligand-dependent transactivation via appropriate reporter constructs.



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Figure 1. mRXR{alpha} and hER Selectively Bind a 65-kDa Protein (p65) in GH4C1 Nuclear Extracts

Nuclear extracts from rat GH4C1 cells were fractionated using DEAE-Sephadex and CM-Sephadex and incubated with agarose beads containing the indicated GST-receptor proteins. Washed beads were analyzed by SDS-PAGE and Coomassie blue staining to detect nuclear proteins interacting with various receptor domains. Panel A, Structure of mRXR{alpha} and hER showing the location of various receptor domains. Panel B, Binding of nuclear extract (N.E.) proteins to various GST-mRXR{alpha} fusion proteins in PBB with 150 mM KCl. Panel C, Binding of nuclear extract proteins to GST-mRXR{alpha}-1–239 and GST-mRXR{alpha}-140–240 in PBB with 75 mM KCl. Extract 1 contained proteins extracted from nuclei with 280 mM KCl. The nuclear pellet was then subjected to a second extraction at 400 mM KCl to yield extract 2. Extracts 1 and 2 were both fractionated on DEAE- and CM-Sephadex before use in the protein-binding experiment. The positions of 50-kDa (p50) and 105-kDa (p105) proteins binding GST-RXR in this experiment are also indicated. Panel D, Binding of nuclear extract proteins to various GST-hER proteins in PBB with 150 mM KCl.

 
N-Terminal receptor domains (A and B) are quite variable and contain a second transactivation region (AF-1) in some receptors (2, 4). Ligand binding was suggested to relieve LBD repression of AF-1 activity because LBD deletion mutants of the glucocorticoid receptor (GR), estrogen receptor (ER), and androgen receptor exhibit constitutive activity (9, 10, 11, 12, 13, 14, 15). Additional evidence that AF-1 function is ligand dependent comes from studies with the antiestrogens ICI 182,780 or 164,384, which can elicit ER binding to DNA without activating transcription (16, 17, 18, 19). It thus appears that AF-1 is repressed by the LBD, and ligand acts, in part, to relieve such repression. This model also implies allosteric interactions between the LBD and N-terminal receptor domains.

DNA binding is mediated by an internal domain (C) containing two Cys4 zinc fingers that bind DNA at HREs composed of two or more hexanucleotide half-sites oriented as inverted, everted, or direct repeats (20, 21, 22). The 68-amino acid DBD is the most highly conserved region of the nuclear receptor family and determines HRE preference and target gene selectivity. For some receptors, the DBD may also participate in receptor dimerization and transcriptional regulation (23, 24). With regard to the latter function, specific DBD deletions or mutations of steroid receptors have been identified that do not prevent DNA binding, yet greatly impair transcriptional regulation (9, 10, 11, 12, 23, 24, 25).

Transactivation by nuclear receptors likely involves the recruitment and stabilization of transcription complexes at target gene promoter sites (2, 4). The precise mechanisms by which nuclear receptors elicit such effects have not been fully defined. However, evidence suggests that coregulators may couple receptors to the transcription complex. For example, ligand-dependent activation by the ER can inhibit transactivation by progestin receptors (26). Such "squelching" implies the existence of a limited supply of one or more coregulators mediating receptor effects. Several laboratories have reported interactions between the LBD of receptors and proteins thought to act as coregulators (27, 28, 29, 30, 31). The highly conserved DBD of nuclear receptors could also serve as a site of interactions for coregulator proteins. We now report the identification of the nuclear protein TLS (translocated in liposarcoma) (32) [also known as FUS (33)] as a high-affinity binding protein for the DBD regions of retinoid, steroid, and thyroid hormone receptors. TLS is a member of the RNP family of RNA-binding proteins and was originally identified as part of a fusion protein arising from a reciprocal chromosomal translocation associated with human myxoid liposarcomas (32, 33). In the resulting fusion protein, the N-terminal half of TLS is joined to CHOP, a CAAT/enhancer binding protein (C/EBP)-related transcription factor with site-specific DNA-binding activity (34). This fusion protein appears to act as a potent chimeric transforming oncogene and transcription factor due to the association of a strong transactivation domain from TLS with the DNA-binding activity of CHOP (35). Recent studies indicate that certain RNP proteins may act to couple transcription to RNA processing (36, 37, 38). In addition, TLS, as well as a recently identified putative TAF (hTAFII68) with homology to the TLS RNP domain, have been shown to be associated with a subpopulation of transcription factor IID (TFIID) complexes (39). These properties of TLS, together with the nature of its interaction with nuclear receptors in vitro and in vivo, suggest a role for TLS in nuclear receptor function and raise the possibility of coupled transcription and RNA processing.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A 65-kDa Nuclear Protein (p65) Interacts with the DNA-Binding Region of Nuclear Hormone Receptors
Glutathione-S-transferase (GST)-receptor fusion proteins bound to glutathione-agarose were incubated with nuclear extracts or ion exchange column fractions, the beads were washed, and bound nuclear proteins were separated by SDS-PAGE and stained with Coomassie blue. Assuming a stoichiometric interaction between potential protein targets and the GST-receptor, it should be possible to isolate and sequence low abundance nuclear proteins (0.01% of total) from as little as 40–50 mg of nuclear protein. Similarly, Coomassie blue staining can detect the binding of as little as 50 ng of a nuclear protein to a GST-receptor protein.

Initial experiments used GST fused to mouse retinoid X receptor-{alpha}1 (mRXR{alpha}) and unfractionated nuclear extracts from rat pituitary GH4C1 cells or HeLa cells that show high levels of stimulation by nuclear hormone receptors (5, 6, 40, 41). Although many extracts revealed the same highly specific interactions with a few select proteins, some nuclear extracts bound GST-mRXR{alpha} in an apparently nonspecific fashion. To reduce nonspecific interactions and improve reproducibility, GST fusion proteins were constructed containing discrete portions of mRXR{alpha}, and nuclear extracts were fractionated on diethylaminoethyl (DEAE)- and carboxymethyl (CM)-Sephadex (Fig. 1Go). GH4C1 nuclear extracts contained an RXR-interacting 65-kDa protein (p65) that passed through DEAE-Sephadex but bound CM-Sephadex at 50 mM KCl and could be eluted from CM-Sephadex at 250 mM KCl. At 150 mM KCl, p65 specifically bound to GST-mRXR{alpha} containing the DBD (amino acids 140–205)(Fig. 1BGo). In particular, GST-mRXR{alpha}-1–467, 1–205, 1–239, and 140–240 all reproducibly bound p65, whereas no interaction was obtained with the LBD (mRXR{alpha}-206–467) or GST alone. GST-mRXR{alpha}-1–239 bound more p65 than either mRXR{alpha}-1–205 or 140–240, suggesting that the N-terminal A/B domain and a small region C-terminal to the DBD stabilized the interaction between the DBD and p65. When the KCl concentration was reduced from 150 mM to 75 mM during protein-binding experiments, GST-mRXR{alpha}-1–239 binding to p65 was little changed, whereas p65-binding by GST-mRXR{alpha}-140–240 now approached that of GST-mRXR{alpha}-1–239 (Fig. 1CGo). In this experiment with 75 mM KCl, smaller amounts of 50-kDa (p50) and 105-kDa (p105) proteins also selectively bound to the GST-RXRs.

The selective binding of p65 to GST-mRXR{alpha} was reproducibly detected using CM-Sephadex eluates from four different preparations of GH4C1 nuclear extract. In one experiment, nuclei that had been extracted with 280 mM KCl were extracted a second time with 400 mM KCl (both extracts were processed using DEAE- and CM-Sephadex). The second extraction at 400 mM KCl yielded little additional p65 or other proteins capable of interacting with GST-mRXR{alpha}-1–239 or GST-mRXR{alpha}-140–240 (Fig. 1CGo). Addition of the RXR ligand 9-cis-retinoic acid (10 µM) during incubations of GST-mRXR{alpha}-1–467 with nuclear extract at 100 mM KCl did not alter the binding of p65 and did not elicit a notable change in the binding of other nuclear proteins (data not shown). The lack of a ligand effect on p65 binding to mRXR{alpha} was not surprising since the interaction is centered on the receptor DBD rather than the LBD. Protein-binding experiments using GST fusions with human ER (hER) indicated that the hER DBD (amino acids 185–250) also bound p65 in GH4C1 nuclear extracts. In buffer containing 150 mM KCl, GST-hER-1–282 bound p65 whereas GST alone or GST-hER-1–185 or GST-hER-278–595, which do not contain the DBD, did not (Fig. 1DGo).

Figure 2Go compares studies with extracts of HeLa cells and rat GH4C1 cells. GST-mRXR{alpha} proteins containing the DBD bound p65 as well as two other proteins (p50 and p105) in both extracts. In multiple experiments the ratio of p65 to p50 or p105 varied suggesting that the binding of p65 to GST-mRXR{alpha} occurred independently of the binding of p50 and p105. HeLa-derived p65 migrated as a slightly larger protein than p65 from rat GH4C1 cells (less than 5-kDa apparent size difference) (Fig. 2Go). Inclusion of a 5-fold molar excess of a palindromic T3 response element (TREp) relative to GST-mRXR{alpha}-140–240 during incubation with nuclear extract had little ef-fect on GST-mRXR{alpha}-140–240 binding to p65, and GST-mRXR{alpha}-140–240 strongly binds the TREp under such conditions (data not shown). Thus, DNA binding does not appear to block the binding of p65 to GST-mRXR{alpha}-140–240. Indeed, even a 1000-fold molar excess of the TREp did not disrupt binding of p65.



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Figure 2. p65 from Nuclear Extracts of GH4C1 Cells and HeLa Cells Interact with GST-mRXR{alpha} Proteins Containing the DBD

Nuclear extracts from GH4C1 cells or HeLa cells were fractionated using DEAE- and CM-Sephadex and incubated with the indicated GST-mRXR{alpha} fusion proteins in PBB with 75 mM KCl. Washed beads were analyzed by SDS-PAGE and Coomassie blue staining. Panel A, Binding of GH4C1 nuclear proteins to various GST-mRXR{alpha} proteins. Panel B, Binding of HeLa nuclear proteins to various GST-mRXR{alpha} proteins.

 
Identification of p65 as TLS
GST-mRXR{alpha}-140–240 is a 36-kDa protein that is well separated in gels from regions containing target proteins of interest for sequencing analysis. p65 bound to GST-mRXR{alpha}-140–240 at 75 mM KCl was not removed by extensive washing at 100 mM KCl but could be selectively eluted at 500 mM KCl (Fig. 3AGo). The eluted material could be recovered and concentrated by ultrafiltration to yield a highly purified protein (Fig. 3BGo), but losses were greater than 50%. Nonetheless, the marked selectivity of GST-mRXR{alpha}-140–240 for p65 indicates that this approach can be used for the large-scale isolation of the protein in its native state from nuclear extracts.



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Figure 3. Purification and Sequencing of p65

Nuclear extracts from GH4C1 cells fractionated using DEAE- and CM-Sephadex were incubated with GST-mRXR{alpha}-140–240 in PBB with 75 mM KCl. Beads were then washed with PBB containing 100 mM KCl or 500 mM KCl. Washed beads were analyzed by SDS-PAGE and Coomassie blue staining. Nuclear extract fractions and concentrated proteins eluting from GST-RXR beads in the 500 mM KCl wash were also analyzed. The p65 bound to GST-mRXR{alpha}-140–240 was cut from stained gels and sequenced as described in the text. Panel A, SDS gel showing proteins bound to GST-RXR after incubation with nuclear extract (N.E.) and washing with 100 mM KCl or 500 mM KCl. Panel B, SDS-gel showing nuclear proteins present in unfractionated extract (CRUDE), DEAE flow-through fraction (DEAE-FT), 250 mM KCl eluate of CM-Sephadex (CM-eluate), and purified nuclear protein eluting from GST-RXR beads in the 500 mM KCl wash (GST-RXR PURIFIED). Proteins in the 500 mM KCl wash of GST-RXR beads that had not been incubated with nuclear extract are also shown (GST-RXR BLANK). Panel C, Sequence of two peptides generated by endoproteinase Glu-C hydrolysis of p65 (peptides 1 and 2) and their alignment with human TLS (32, 33). Note that both peptides are C-terminal to a Glu residue (E, an endoproteinase Glu-C cleavage site).

 
For sequence analysis of p65, 30 mg of GH4C1 nuclear protein were fractionated using DEAE-Sephadex and CM-Sephadex, and the final CM-Sephadex elute was used in protein-binding experiments with GST-mRXR{alpha}-140–240. After SDS-PAGE and Coomassie blue staining, gel slices containing a total of 5–6 µg of p65 were electroeluted in the presence of endoproteinase Glu-C, and the cleaved peptides were resolved by HPLC. Two of the peptides were sequenced (Fig. 3CGo) and were found to be 100% identical to amino acids 300–310 and 338–348 of human TLS (32, 33). Human TLS is a 526-amino acid protein with a predicted mass of 53,370 that migrates anomolously in SDS gels as a 65- to 68- kDa protein (32), in agreement with the approximate molecular mass of 65 kDa assigned to the RXR-interacting protein.

Western blot analysis using a monoclonal antibody against the C-terminal region of human TLS (32) was used to confirm the identity of p65 as TLS. Using unfractionated GH4C1 nuclear extracts in 150 mM KCl, GST-mRXR{alpha}-1–467, 1–239, and 140–240 bound a TLS-immunoreactive protein of 65 kDa, whereas GST alone and GST-mRXR{alpha}-206–467 did not (Fig. 4AGo). The amount of immunoreactive TLS bound by the various GST-mRXR{alpha} proteins paralleled the amounts of p65 bound as detected by Coomassie blue staining (see Fig. 1BGo). Similarly, GST-hER-1–287 and GST fused to chicken thy-roid hormone receptor-{alpha} (cTR{alpha}-1–408) bound immunoreactive TLS from GH4C1 cells while GST-hER-1–185 did not (Fig. 4BGo). Finally, GSTmRXR{alpha}-1–239 and GST-hER-1–287 bound immunoreactive TLS from unfractionated HeLa nuclear extracts, whereas GST-mRXR{alpha}-206–467 and GST-hER-1–185 did not (Fig. 4CGo). The Western blot data confirm that TLS from rat GH4C1 cells and human HeLa cells strongly interacts with GST-receptor fusions containing the DBD of either retinoid, thyroid hormone, or estrogen receptors.



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Figure 4. GST-Receptor Proteins Bind Immunoreactive-TLS in Unfractionated Nuclear Extracts from GH4C1 Cells and HeLa Cells

Unfractionated nuclear extracts were incubated with agarose beads containing the indicated fusion proteins in PBB with 150 mM KCl. Western blot analysis of washed beads used a monoclonal antibody against the human TLS C-terminal domain. Panel A, Binding of TLS in GH4C1 nuclear extracts by various GST-mRXR{alpha} proteins. Panel B, Binding of TLS in GH4C1 nuclear extracts by various GST fusions with mRXR{alpha}, hER, and cTR{alpha}. Panel C, Binding of TLS in HeLa cell nuclear extracts by GST fusions with mRXR{alpha} and hER.

 
Interaction of TLS with cTR{alpha}
To further analyze TLS interactions with receptors, GST fusions with human TLS-1–274 (TLS-N) and TLS-275–526 (TLS-C) were used to assess interactions with various domains of [35S]cTR{alpha} synthesized in vitro (Fig. 5Go). TLS-1–274 corresponds to the TLS region that yields a potent transactivation factor and transforming oncogene when fused to CHOP (35). This TLS region contains an N-terminal domain rich in glutamine, serine, and tyrosine [amino acids (aa) 1–165] as well as a glycine-rich domain containing five poly-Gly tracts and six Arg-Gly-Gly repeats (aa 166–274)(32). TLS-275–526 contains a putative RNA-recognition (RNP) motif (aa 287–372) and a second Gly-rich domain with four poly-Gly tracts and 13 Arg-Gly-Gly repeats.



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Figure 5. Binding of Various cTR{alpha} Domains to the N-Terminal and C-Terminal Regions of TLS

The indicated cTR{alpha} domains encoded in pEXPRESS vectors were synthesized and labeled with [35S]cysteine using in vitro transcription and translation with reticulocyte lysate and T7 polymerase. [35S]cTR{alpha} products were incubated with agarose beads containing GST alone, GST-TLS-1–274 (GST-TLS-N), or GST-TLS-275–526 (GST-TLS-C) in PBB containing 100 mM KCl. Washed beads were analyzed by SDS-PAGE followed by fluorography of gels dried in 8% sodium salicylate. Panel A, cTR{alpha} receptor domains. Panel B, Fluorographs showing [35S]cTR{alpha} domains bound to the indicated GST fusion proteins; one tenth of [35S]cTR{alpha} inputs during incubations with GST-fusions are also shown.

 
At 100 mM KCl, GST alone failed to interact with any [35S]cTR{alpha} protein. GST-TLS-1–274, however, bound [35S]cTR{alpha}-1–408 and 1–151 with high affinity. In contrast, GST-TLS-275–526 exhibited a much lower affinity for these cTR{alpha} proteins. Both GST-TLS proteins bound [35S]cTR{alpha}1–118 and 51–154 with low affinity (binding of GST-TLS-275–526 to [35S]cTR{alpha}1–118 was evident in autoradiographs but was too weak to detect in photographic reproductions). Neither GST-TLS protein bound [35S]cTR{alpha}-120–408 (which includes the entire LBD) (Fig. 5BGo). These data suggest that TLS-1–274 binding to cTR{alpha} is centered around the DBD (aa 51–118) with the immediate flanking regions also participating in the interaction. The finding that both GST-TLS proteins exhibited a similar low affinity for [35S]cTR{alpha}-1–118 and 51–154 indicates that cTR{alpha} regions 1–51 and 119–151 do not appear to contribute to TLS-275–526 interactions with [35S]cTR{alpha}. Overall, the ability of both TLS-1–274 and 275–526 to bind [35S]cTR{alpha}, and the varying strengths of their interactions, suggests a potential role for the poly-Gly TLS domains in [35S]cTR{alpha} binding.

T3 did not alter the affinity of [35S]cTR{alpha} for either GST-TLS-1–274 or 275–526 at KCl concentrations of 100 or 200 mM (Fig. 6AGo). The strength of the interaction of GST-TLS-1–274 with 35S-cTR{alpha} was further assessed by conducting the binding reaction for 60 min at 250 mM KCl, followed by an incubation with varying KCl concentrations for 30 min. KCl concentrations as high as 1 M at pH 7.9 or 700 mM at pH 6.0 (to partially neutralize acidic residues in cTR{alpha}) did not dissociate [35S]cTR{alpha} bound to GST-TLS-1–274 (Fig. 6BGo), indicating a stable high-affinity complex.



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Figure 6. Effect of T3 and KCl Concentrations on the Binding of cTR{alpha} to GST-TLS-N and GST-TLS-C

[35S]-cTR{alpha}-1–408 was incubated with GST-TLS-1–274 (GST-TLS-N) or GST-TLS-275–526 (GST-TLS-C) in the presence or absence of 5 µM T3 in PBB containing the indicated KCl concentrations. [35S]cTR{alpha} bound to the washed beads was electrophoresed in SDS gels, and the [35S]cTR{alpha} was detected by fluorography. Panel A, [35S]cTR{alpha} bound to GST-TLS fusion proteins after incubation in the presence or absence of T3 in PBB containing 100 or 200 mM KCl. Panel B, [35S]cTR{alpha} bound to GST-TLS-N after incubation for 60 min in PBB containing 250 mM KCl, followed by a 30-min wash with PBB containing the indicated KCl concentration at either pH 7.9 or pH 6. All beads were rinsed with PBB containing 100 mM KCl after the first wash to equalize salt loads in the samples during electrophoresis.

 
The affinity and KCl stability of [35S]cTR{alpha} binding to GST-TLS-1–274 was compared with that for GST-mRXR{alpha} proteins because RXR is known to form a high-affinity interaction with TR (42, 43, 44) (Fig. 7Go). Remarkably, the affinity of cTR{alpha} for GST-TLS-N-1–274 was greater than for GST-mRXR{alpha}-206–467, which comprises the LBD of mRXR{alpha} and contains an essential region required for the formation of TR-RXR heterodimers (43, 44). GST-mRXR{alpha}-1–239 also bound [35S]cTR{alpha}, and the interaction was only slightly weaker than that found with GST-mRXR{alpha}-206–467 (Fig. 7Go). This result was of interest because the DBD of RXR has been reported to contribute to the formation of TR-RXR heterodimers on direct repeat DNA elements (45, 46, 47). A comparison of the binding of GST-mRXR{alpha}-1–467, GST-mRXR{alpha}-1–239, and GST-mRXR{alpha}-206–467 for cTR{alpha} indicated that the N-terminal half of mRXR{alpha} (aa 1–239) and the LBD (aa 206–467) synergize to enable the formation of stable, high-affinity cTR{alpha}-mRXR{alpha} heterodimers in solution. The salt stability of cTR{alpha}-TLS-N complexes was similar to that of cTR{alpha}-mRXR{alpha}-1–467 complexes. Thus, the affinity of TLS for cTR{alpha} is similar to mRXR{alpha}, a known physiological binding partner for cTR{alpha}.



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Figure 7. Comparison of [35S]cTR{alpha} Binding to GST-TLS-1–274 and GST-mRXR{alpha}

[35S]cTR{alpha}-1–408 was incubated with GST-TLS-1–274 (GST-TLS-N) and the indicated GST-mRXR{alpha} proteins were incubated in PBB with the indicated KCl concentrations. Washed beads were analyzed by SDS-PAGE and fluorography. The figure compares the binding of [35S]cTR{alpha} to GST-TLS-N, GST-mRXR{alpha}-1–467, 1–239, or 206–467.

 
TLS-1–274 Interacts with the DNA-Binding Region of the Rat GR (rGR)
mRXR{alpha}, cTR{alpha}, and hER are members of the receptor superfamily that bind to HREs containing consensus AGGTCA half-sites. Binding of the rGR DBD by TLS was evaluated to determine whether receptors with differing HRE half-site specificity (AGAACA for the rGR) could also interact with TLS. Recombinant rGR-440–525 protein (encoding the DBD and 24 additional residues toward the C terminus) bound GST-TLS-1–274 at 50, 100, or 200 mM KCl. GST alone did not bind rGR-440–525 (Fig. 8Go). At 100 mM KCl, 20 pmol (1.5 µg) GST-TLS-1–274 bound approximately 16 pmol (240 ng by scanning densitometry) of the 1.4-µg rGR-440–525 input. This is a near-stoichiometric interaction of rGR-440–525 with GST-TLS-1–274. Thus, TLS appears to display high affinity for members of the nuclear receptor family with widely varying ligand and HRE specificities.



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Figure 8. The rGR DNA-Binding Region Binds to GST-TLS-1–274

Purified rGR-440–525 (rGR: 1.4 µg) was incubated with agarose beads containing 1.5 µg of GST alone or GST-TLS-1–274 (GST-TLS-N) in PBB containing the indicated KCl concentrations. Washed beads were analyzed by SDS-PAGE and Coomassie blue staining. rGR-440–525 binding to GST-TLS-N was quantitated by scanning densitometry. Total rGR-DBD input during incubations with the GST proteins is also shown (INPUT). Protein bands corresponding to GST alone and GST-TLS-N were present above the gel region shown.

 
cTR{alpha} Can Bind DNA Response Elements While Bound to TLS
TLS contains a transactivation domain in its N terminus that appears to be critical for the transforming activity of the TLS-CHOP fusion protein (35). However, the functional role of the wild-type TLS is unclear. The interaction of TLS with members of the nuclear receptor family suggests that wild-type TLS may be targeted to specific gene promoters through its interaction with nuclear receptors. For this to occur, nuclear receptors bound to TLS should also be capable of binding to their specific HREs. The finding that high concentrations of the TREp do not block the binding of TLS to GST-mRXR{alpha}-140–240 suggests this possibility. To study this more directly, we examined the ability of cTR{alpha} to interact with 32P-labeled DNA encoding a DR4 HRE ([32P]DR4) while tethered to GST-TLS-1–274 (Fig. 9Go). Agarose beads containing GST alone, GST-TLS-1–274, or GST preincubated with cTR{alpha} did not bind significant amounts of [32P]DR4. However, preincubation of GST-TLS-1–274 with soluble cTR{alpha} led to a striking 24-fold increase in the amount of [32P]DR4 bound by GST-TLS-1–274. Addition of T3 during the incubations did not alter the amount of [32P]DR4 bound. Thus, it appears that the interaction of TLS with members of the nuclear receptor family may enable TLS to be targeted to specific gene promoters in concert with the nuclear receptor.



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Figure 9. cTR{alpha} Binds a DR4 HRE While Bound to TLS-1–274

Agarose beads containing GST alone or GST-TLS-1–274 were incubated with or without purified cTR{alpha} in PBB with 100 mM KCl, washed three times, and then incubated with a 32P-labeled DR4 HRE. After washing, the beads were counted for 32P. In some reactions, T3 was included at a concentration of 5 µM in both incubation and wash buffers. [32P]DR4 input = 39,300 cpm.

 
Physical and Functional Interactions of cTR{alpha} with TLS
Several approaches were used to identify an interaction of cTR{alpha} and TLS in the cell. Immune precipitation of cell lysates was not revealing. However, we found that various anti-cTR{alpha} antibodies did not immunoprecipitate cTR{alpha}-TLS complexes in vitro. In addition, these antibodies blocked the interaction of [35S]cTR{alpha} with GST-TLS-N or [35S]TLS with GST-cTR{alpha} in vitro, suggesting that TLS interferes with antibody recognition of the complex. Thus, we used pEBG-cTR{alpha}-1–408, a mammalian GST-cTR{alpha}-1–408 expression vector, to provide evidence for TLS binding after expression of receptor in cells. 293T cells were used in these studies since the promoter in the pEBG vector has been shown to express GST-fusion proteins in these cells (48). In transfection studies, GST-cTR{alpha}-1–408 was found to be as active as wild-type cTR{alpha}-1–408 in mediating basal repression or ligand-dependent activation of {Delta}MTV-TREp-CAT. For analysis of TLS interactions, cells were transfected with pEBG-cTR{alpha}-1–408 or the pEBG GST control vector and 40 h later T3 was added to half of the flasks for 1 h. The cells were then harvested and the cytosol and nuclear fractions prepared by the method of Dignam et al. (49). Unfractionated nuclear extracts and cytosol were incubated with glutathione-agarose for 1 h at 4 C. The beads were then washed, boiled in SDS loading buffer, and analyzed for GST or TLS by Western blotting. Figure 10AGo shows that nuclear extracts contain similar amounts of GST or GST-cTR{alpha}-1–408 after transfection with the indicated pEBG vectors. However, GSH-agarose binding only detected immunoreactive TLS in the nuclear extracts of cells expressing GST-cTR{alpha}-1–408 (Fig. 10BGo). As with the in vitro binding studies, T3 did not alter the TLS-cTR{alpha} interaction. GST immunoreactivity was also present in the cytosolic fraction. However, GST-cTR{alpha}-1–408 was not detected, and no TLS was identified in the binding assay (not shown). Although these studies support the notion that cTR{alpha} and TLS can interact in vivo, we cannot exclude the possibility that TLS and GST-cTR{alpha}-1–408 associate in vitro after the nuclear extraction.



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Figure 10. Binding of TLS after Expression of Thyroid Hormone Receptor in Cells

Nuclear extracts from 293T cells expressing GST or GST-TR were incubated with GSH agarose beads as described in Materials and Methods. A, Western blot with antibody against GST indicating the relative levels of expression of GST and GST-TR fusion protein in the transfected cells. B, Western blot with antibody against TLS indicating that TLS associates with GST-TR but not with GST alone. N.E. is nuclear extract from 293T cells that was used as a standard to identify the electrophoretic migration of TLS. Lanes 1, 2, 3, and 4 are duplicate experiments analyzed in the same gel.

 
Since the N-terminal half of TLS interacts with the region of cTR{alpha} containing the DBD in vitro, we sought to determine whether this interaction could occur in the intact cell. Transfection studies were performed with pcDNA vectors expressing wild-type TLS, TLS-N, and TLS-C using CaPO4 coprecipitation. These studies were performed in an effort to identify a dominant negative effect or other evidence for a functional interaction of cTR{alpha} with TLS. Because TLS is a highly abundant protein, we used 30 µg of these expression vectors to transfect HeLa cells or 293T cells to express these proteins at levels higher than the endogenously expressed TLS. Although we could not express TLS at levels higher than endogenous TLS, we were able to express TLS-N or TLS-C at levels that were at least 10-fold greater than endogenous TLS (established by Western blotting). In those experiments (in the absence of cTR{alpha}), TLS expression mediated a modest reduction in basal gene activity, TLS-C had no effect, and TLS-N markedly increased expression (~20-fold) of {Delta}MTV-TREp-CAT or {Delta}SV-DR4-CAT. Similar results were also found with Rat2 fibroblasts. The mechanism of promoter stimulation mediated by TLS-N was not established but may reflect its association with basal transcription factors when expressed at very high levels. This high level of promoter activity evoked by TLS-N precluded its use as a dominant negative probe for analysis of TLS-cTR{alpha} interactions in vivo. Expression of TLS-C did not alter T3-dependent stimulation by cTR{alpha}.

Because of the marked stimulation mediated by expression of high levels of TLS-N, we used a mammalian two-hybrid approach to provide evidence for a TLS-receptor interaction (Table 1Go). GAL4-TLS-N is known to be a potent transactivator (35). An amount of GAL4-TLS-N vector was used (0.2 µg) that gives moderate stimulation to assess whether the various cTR{alpha} proteins could interact with GAL4-TLS-N in vivo and, thus, alter its activity. Transfection of HeLa cells with GAL4-TLS-N, but not the GAL4-DBD, activated gene expression from pMC110, a GAL4-CAT reporter gene. Expression of cTR{alpha}(120–408), containing only the LBD, did not alter the extent of stimulation by GAL4-TLS-N (Table 1Go). In contrast, wild-type cTR{alpha}(1–408) repressed activation by GAL4-TLS-N, and this repression was largely reversed by T3. This finding is consistent with the notion that cTR{alpha} interacts with TLS via its DBD and that a transcriptional inhibitor(s) binds to the LBD of TR and is released by T3 to unmask the TLS-dependent transactivation (50). Two recently cloned factors (SMRT and NcoR) are candidates for this corepressor activity (51, 52). Additional evidence to support an interaction of the N-terminal region of cTR{alpha} containing the DBD with the N terminus of TLS comes from studies with cTR{alpha}(1–221)VP16. This chimera lacks most of the LBD and, therefore, would not be expected to associate with transcriptional repressor proteins. Thus, an association of cTR{alpha}(1–221)VP16 with GAL4-TLS-N would not result in repression but would be expected to lead to further activation via the VP16 activation domain. Table 1Go shows that expression of cTR{alpha}(1–221)VP16 does not alter the activity of the GAL4-DBD but further enhances activation by GAL4-TLS-N, providing further evidence that cTR{alpha} and TLS can interact in the cell.


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Table 1. Functional Interactions of TLS with cTR{alpha}

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TLS Interacts with the DNA-Binding Region of Nuclear Hormone Receptors
We have identified TLS as a nuclear protein that binds members of the nuclear receptor family with high affinity in a ligand-independent manner. TLS binding to various GST-receptor domains, and binding of cTR{alpha} deletion mutants to GST-TLS, involves the receptor DBD with closely flanking regions contributing to the stability of the complex. TLS binds to nuclear receptors primarily through its N-terminal half. Experiments with full-length [35S]cTR{alpha} indicated that TLS binding is stable in 1 M KCl (Fig. 6Go); thus, TLS binding is unlikely to reflect a simple ionic interaction with acidic elements of receptor DBDs. Indeed, the C-terminal half of TLS, containing more than 75% of its basic residues and its most hydrophilic domains, displayed low affinity for cTR{alpha}. TLS binding to nuclear receptors also displayed high specificity. Only two other proteins (p50 and p105) exhibited binding to GST-mRXRß approaching that of TLS. Studies are in progress to identify and/or clone p50 and p105, which do not react with TLS-monoclonal antibody (Fig. 4Go) or polyclonal antibody (not shown). Finally, TLS from either rat or human cell lines bound nuclear receptors from diverse species (mouse, chicken, human, and rat). Thus, the interaction between TLS and nuclear receptors appears to have been highly conserved among vertebrates.

TLS is one of only a few proteins identified to interact with the DNA-binding region of either steroid, thyroid, or retinoid receptors without interfering with DNA binding. Calreticulin has been reported to interact with the DBD of the GR and interfere with receptor activity when expressed in vivo (53). c-Jun and the p53 tumor suppressor have been reported to decrease TR DNA binding and transactivation via interaction with the DBD (54, 55). In contrast, like TLS, the human immunodeficiency virus type 1 (HIV-1) tat transactivator binds the TR DBD without altering DNA binding (56), and this interaction results in enhanced T3 stimulation of the HIV-1 long terminal repeat (56, 57). The structural conservation of the DBD of nuclear receptors may allow for an interaction of diverse members of the receptor family with a common binding protein (i.e. TLS). However, the regions flanking the DBD in the TRs and RXRs also appeared to contribute to TLS binding. Unlike the DBD, these flanking regions are quite variable among nuclear receptors but may nonetheless possess common structural features affecting DBD conformation and TLS binding.

TLS (32) [also known as FUS (33)] was originally identified as part of a fusion protein arising from a chromosomal translocation in human myxoid liposarcomas (32, 33). In this protein, the N-terminal half of TLS is fused with the open reading frame of CHOP, a member of the C/EBP family of transcription factors. In certain human myeloid leukemias, the N-terminal half of TLS is fused with the DBD of ERG (a member of the ets family of transcription factors) (58). Human TLS is a 526-residue protein that is closely related to EWS (656 residues, 55.6% identity) (32). The N-terminal half of EWS has been identified in translocation-induced fusions with different transcription factors in Ewing sarcomas (FLI-1, ERG, ETV-1) (59, 60, 61), a C/EBP family member (ATFI) in malignant melanoma of soft parts (62), the Wilm’s tumor gene product (WT1) in desmoplastic small round cell tumors (63), and an orphan member of the steroid/thyroid receptor family (CHN) in myxoid chondrosarcoma (64). A feature of all tumor-derived TLS and EWS fusion proteins is the fusion of the N-terminal half of TLS or EWS with transcription factor domains containing site-specific DNA-binding activity.

The functional role(s) of wild-type TLS or EWS remains to be determined. However, analysis of TLS and EWS fusion proteins indicate that their N-terminal regions possess a potent transactivation domain rich in Gln, Tyr, and Ser residues (35, 65, 66, 67). It appears that fusion of such domains with transcription factor DBDs leads to aberrant transcriptional regulation and cell transformation. In view of such findings, the identification of TLS as a high-affinity binding protein for the nuclear receptor family of transcription factors suggests a role for TLS in nuclear receptor function. In particular, in vitro studies with GST-TLS-1–274 revealed that TLS-1–274 lacks intrinsic affinity for a DR4 HRE but can associate with the HRE via a TLS-cTR{alpha} complex. Thus, TLS binding to nuclear receptors may tether its transactivation domain to specific gene promoters where further interactions may occur relevant to transcriptional regulation. Consistent with this model are studies indicating that TLS and cTR{alpha} may physically and functionally interact after expression in vivo (Fig. 10Go and Table 1Go). Because of the high abundance of TLS, a more definitive analysis of functional TLS-cTR{alpha} interactions will require the use of TLS-deficient cell lines and/or TLS knockout mice.

TLS Is a Member of a Nuclear RNA-Binding Protein Family That May Act to Couple Transcriptional Activation with RNA Processing
TLS and EWS appear to comprise a distinct subfamily of RNP proteins (32, 33, 35, 59) that has been highly conserved through evolution because a closely related gene (SARFH) is expressed in Drosophila (68, 69). Like other RNP proteins (70), TLS and EWS contain a RNP domain and two regions with multiple ArgGlyGly repeats and bind RNA in vitro (32, 68, 71). The RNA-binding activity of TLS is primarily contributed by its C-terminal half containing the RNP domain and numerous ArgGlyGly repeats (32). Thus, nuclear receptor binding and RNA binding appear to be mediated by separate domains. In view of TLS and EWS similarities, it is notable that Western blot analysis did not reveal EWS binding to GST-receptor fusions even though substantial EWS was present in nuclear extracts (data not shown). GST-receptor fusions also did not bind hnRNP-Al, an abundant RNP protein with features reminiscent of TLS. Thus, TLS may be unique among known RNP proteins in its high affinity for nuclear receptors.

TLS and SARFH can be detected in intimate association with transcriptionally active chromatin and have been suggested to participate in transcriptional regulation and/or hnRNA processing in concert with other RNP proteins (35, 68, 72). In addition, TLS, as well as a recently identified putative TAF (hTAFII68) with homology to the TLS RNP domain, have been shown to be associated with a subpopulation of TFIID complexes (39). These findings suggest that TLS binding to nuclear receptors might serve as a priming mechanism to recruit TLS to the TFIID complex or to accelerate the subsequent processing of primary transcripts generated in response to hormone-induced transcription. This role would be consistent with recent studies indicating that certain RNP proteins may act to couple transcription to RNA processing (36, 37, 38).

Interplay of the A/B domain, DBD, and the LBD in Ligand-Mediated Transcriptional Activation
The present studies were undertaken to identify potential coregulators that may participate in the transcriptional actions of nuclear receptors. The identification of TLS as a potential coregulator is promising in view of the evidence indicating that TLS is localized at transcriptionally active genes (68) and a subset of TFIID complexes (39) and generates a potent transcription factor when fused with disparate DNA-binding proteins. However, our identification of the receptor DNA-binding region as the site of interaction was somewhat unexpected because numerous studies have indicated a role for the LBD in hormone-induced transactivation. Thus, the LBDs of nuclear receptors can effectively mediate ligand-dependent transactivation when coupled to heterologous DBDs derived from transcriptional activators such as GAL4 (6, 8). Nonetheless, N-terminal domains of many nuclear receptors possess a transactivation region (AF-1) distinct from that in the LBD (AF-2). The GR, ER, and androgen receptor become constitutive transactivators after removal of their LBDs, suggesting that the LBD may mask the transcriptional effects of AF-1 in a ligand-dependent manner (9, 10, 11, 12, 14, 15). Many of these studies also suggested a role for the DBD in transcriptional regulation distinct from DNA binding per se. Indeed, studies in yeast have identified mutations in the GR DBD that interrupt transactivation without altering DNA binding (23, 24, 25). Thus, it has been suggested that the DBD and the N-terminal AF-1 domain may interact to mediate some of the transactivation produced by nuclear receptors. In this regard, it is of note that zinc finger DBDs of some other transcription factors have been implicated in a transcriptional role distinct from DNA binding (73, 74, 75).

In the ER, activity of the N-terminal AF-1 domain is ligand-dependent (see Introduction), and DNA binding by retinoic acid receptors can modulate LBD interactions with putative corepressor proteins (76). Such findings imply reciprocal interactions between the LBD, DBD, and N-terminal A/B domains. The interaction of TR and retinoic acid receptors with large corepressor proteins (N-CoR, 270 kDa; SMRT, 168 kDa) (51, 52) involves a region of the LBD that closely flanks the DBD. Ligand binding leads to dissociation of corepressors from these receptors (50, 51, 52), which may permit various coregulators to participate in transactivation. Thus, corepressors may suppress the activity of the receptor-TLS complex, and ligand-evoked dissociation of corepressors from the LBD may relieve this repression. Indeed, actions of wild-type cTR{alpha} to block transactivation mediated by GAL4-TLS-N, and the reversal of this repression by T3 (Table 1Go), are consistent with this model. In some respects, it might be anticipated that the most highly conserved region of the nuclear receptor family (the DBD) may play a role in transcriptional regulation distinct from its role in DNA binding. The structural conservation of the DBD may enable a single coregulator targeted to this domain to couple a wide array of receptors with differing ligands to identical components of the transcription complex. The structural variability among receptor LBDs and N-terminal A/B domains, on the other hand, suggests that a number of coregulators may be required to mediate such transcriptional roles. Identification of TLS as a high-affinity binding protein for nuclear receptors may facilitate efforts to understand the interplay between the DBD and other receptor domains in transcriptional regulation by nuclear receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
pGEX2T-mRXR{alpha}, which expresses a fusion protein between GST and wild-type mRXR{alpha}-1–467, was provided by Paul T. van der Saag (28). GST fusions with deletion mutants of mRXR{alpha} (mRXR{alpha}-1–205, mRXR{alpha}-1–239, mRXR{alpha}-140–240, and mRXR{alpha}-206–467) were constructed by PCR amplification of mRXR{alpha} using 5'-primers containing a BglII site linked to the first codon of mRXR{alpha} in-frame with pGEX2T, and a 3'-primer ending with an in-frame stop codon extended with an EcoRI site. BglII-EcoRI digests of PCR products were cloned into the corresponding sites of BamHI-EcoRI-digested pGEX2T.

pGEX2T-cTR{alpha}-1–408 expresses GST fused with wild-type chicken thyroid hormone receptor {alpha} (cTR{alpha})(41). pGEX vectors expressing GST fusions with regions of the human estrogen receptor (hER) (GST-hER-1–185; GST-hER-1–287; GST-hER-278–595) were provided by Peter J. Kushner (77) or Myles Brown (27). GST linked to the N-terminal and C-terminal regions of human TLS were constructed by David Ron (manuscript submitted). GST-TLS-1–274 was cloned using an artificially created BamHI site in the 5'-untranslated region of TLS cDNA (32) and a unique internal BspHI site in the coding sequence. GST-TLS-275–526 was cloned using the same BspHI site in the coding sequence and an XhoI site at the 3'-end of the TLS cDNA.

pEBG is a vector regulated by the human elongation factor 1-{alpha} promoter that expresses GST in mammalian cells and has been used to express GST-fusion proteins in 293T cells (48). The entire cTR{alpha}-1–408 was excised from pGEX2T-cTR{alpha}-1–408 with BamHI and was cloned into the analogous BamHI site of pEBG to form pEBG-cTR{alpha}-1–408.

pEXPRESS vectors containing the Rous sarcoma virus long terminal repeat linked to a phage T7 RNA polymerase promoter (78) were used for in vitro transcription and translation of cTR{alpha}-1–408, cTR{alpha}-1–118, cTR{alpha}-1–151, cTR{alpha}-51–154, and cTR{alpha}-120–408 (41). cTR{alpha}(1–392)VP16 was constructed from wild-type cTR{alpha} in pEXPRESS by excising the DNA encoding the last 16 amino acids of cTR{alpha} with SacI and AfI III and replacing the DNA with the SacI-AfIIII fragment from GAL4-VP16 (50). This links amino acid 392 of cTR{alpha} in-frame with the 90-amino acid transactivation domain of the Herpes simplex virus VP16 activator. cTR{alpha}(1–221)VP16 was constructed by blunt-end ligation after the NaeI and SacI fragment were deleted from cTR{alpha}(1–392)VP16. This removes most of the LBD of cTR{alpha}. pSG424 expressing the yeast GAL4 DBD and pMC110, a GAL4 chloramphenicol acetyltransferase (CAT) reporter gene, were described previously (79). GAL4-TLS-N contains the N-terminal region (aa 1–267) of TLS linked in-frame to the GAL4-DBD in pSG424 (35).

Experiments with the rGR DBD (rGR-440–500) used a recombinant peptide prepared as previously described from a plasmid encoding the rGR region 440–525 (80).

Preparation of Nuclear Extracts
HeLa or GH4C1 cells were grown to 70–80% confluence in DHAP medium (50, 56) containing 10% calf serum. Cells were harvested using 1.5 mM EDTA in Dulbecco’s PBS. After cell detachment, the EDTA was neutralized with an equal volume of serum-free DHAP medium. All subsequent steps were conducted at 0–5 C using a procedure similar to that of Dignam et al. (49). Cells were pelleted at 500 x g for 10 min and washed once in PBS and once with 20 volumes of hypotonic lysis buffer (10 mM Tris-HCl, pH 7.8, 10 mM KCl, 1 mM MgCl2). The cells were resuspended in four packed-cell volumes of hypotonic lysis buffer and disrupted in a Dounce homogenizer with a loose-fitting ("B") pestle. Nuclei were pelleted at 2000 x g and, after the supernatant was removed, the nuclear pellet was recentrifuged at 10,000 x g for 15 min. The nuclei were resuspended in 2 volumes of nuclear extraction buffer (10 mM Tris-HCl, pH 7.8, 420 mM KCl, 25% glycerol, 1 mM dithiothreitol (DTT), 1 mM PMSF) to yield a final KCl concentration of about 280 mM. Nuclei were then homogenized with a Dounce B-pestle, and the homogenate was gently stirred for 30 min to release soluble proteins from nuclei. After centrifugation at 15,000 x g for 25 min, the supernatant was dialyzed for 3–5 h against 20 mM Tris-HCl, pH 7.8, 100 mM KCl, 25% glycerol, 1 mM DTT, 0.5 mM PMSF. The buffer was changed once. The dialyzed nuclear extract was centrifuged for 15 min at 13,000 x g to remove any precipitate, and the final supernatant was stored as aliquots at -70 C until use. Protein concentrations of nuclear extracts ranged from 3–5 mg/ml for HeLa cells and 6–9 mg/ml for GH4C1 cells.

Nuclear extracts were usually fractionated on DEAE-Sephadex and CM-Sephadex before use in protein-binding assays using GST-fusion proteins. The extracts were diluted in 20 mM Tris-HCl (pH 7.8) containing 10% glycerol and 2 mM DTT to give a final KCl concentration of about 50 mM. After centrifugation (15 min at 13,000 x g in a microfuge), diluted extracts (3–4 mg total protein in 4–5 ml) were applied to microcolumns containing 0.5–1.0 ml of packed DEAE-Sephadex A-50 equilibrated in elution buffer (EB) containing 20 mM Tris-HCl, pH 7.8, 50 mM KCl, 2 mM DTT, and 10% glycerol. The flow-through fraction was collected and combined with a second elution with one column volume of EB. This material was then applied to 0.5- to 1.0-ml columns of CM-Sephadex C-50 equilibrated in EB. After a wash with three to four column volumes of EB, bound proteins were eluted with three column volumes of EB containing 250 mM KCl. This fraction was then diluted with buffer containing 20 mM HEPES (pH 7.9), 10% glycerol, and 2 mM DTT to give the indicated concentration of KCl and adjusted to contain 1 mM MgCl2, 10 µM ZnCl2, 0.05% Triton X-100, and 40 µg/ml leupeptin.

Preparation of GST Fusion Proteins
Escherichia coli expressing GST-fusion proteins was incubated with 1 mM isopropyl-ß-D-thiogalactopyranoside for 25 min at 37 C. Cultures were then chilled in ice for 10 min and centrifuged at 2000 x g for 10 min at 5 C, and the supernatant was discarded. All subsequent procedures were conducted at 0–5 C. Cell pellets from each 500-ml culture were resuspended in 25 ml PBS containing 40 µg/ml leupeptin, 20 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 40 mM EDTA. Cells were incubated with 400 µg/ml lysozyme for 10 min on ice and then disrupted using a cell sonicator (125 watts for 10–15 sec, repeated three times). The samples were then centrifuged at 10,000 x g for 15 min, and the supernatant was then incubated with 300–500 µl of a 1:1 slurry of GSH-agarose beads in PBS for 20 min. The GSH beads were then washed three times in 50 ml PBS: the last wash included 2 mM DTT and 50 µM ZnCl2 to reform any denatured zinc finger domains present in the GST-receptor fusion proteins. The samples were resuspended in an equal volume of glycerol and stored at -20 C until use. Storage in 50% glycerol at -20 C does not alter GST-fusion protein binding to GSH-agarose and prevents proteolytic degradation of fusion proteins that may occur upon storage at 5 C.

Protein-Binding Assays
Nuclear extracts, chromatography fractions, or in vitro transcription and translation products were diluted in protein-binding buffer (PBB) (20 mM HEPES, pH 7.9, 100 mM KCl, 1 mM MgCl2, 10 µM ZnCl2, 2 mM DTT, 10% glycerol, 0.05% Triton X-100, 40 µg/ml leupeptin). In some experiments, KCl concentrations were varied as noted. In protein-binding assays using nuclear extracts or purified rGR-DBD, 1–2 µg of GST-fusion protein bound to GSH-agarose (7.5–15 µl) were used. In experiments comparing the protein-binding efficiencies of different GST-proteins, GSH-agarose was added as needed to yield equivalent pellet volumes. The samples were rotated for 1 h at 5 C with 1 ml of nuclear extract (300–750 µg protein/ml). Pellets were centrifuged at 1000 x g for 4 min, washed two or three times with 1 ml cold PBB, and then stored at -20 C until analysis by SDS-PAGE. The samples were then suspended in 2–3 volumes of Laemmli sample buffer containing 100 mM DTT, heated for 5 min at 100 C, and electrophoresed in 10% or 12% SDS-polyacrylamide gels as appropriate. In some experiments, nuclear extracts were incubated with GST-RXR proteins with a palindromic HRE (TREp) for TRs and RXRs (5'AGGTCA TGACCT-3') with HindIII cohesive ends.

Protein-binding experiments with 35S-labeled proteins labeled in reticulocyte lysates used 400–500 ng GST-fusion protein in a packed agarose volume of 5–6 µl. L-[35S]cysteine-labeled cTR{alpha} proteins were prepared using 2 µg of appropriate pEXPRESS vectors and TNT reticulocyte lysates (Promega, Madison, WI)(41). Approximately 1 µl of 35S-labeled reaction product (from a total of 50 µl) was used in each binding assay. The incubations with reticulocyte lysate-labeled proteins also contained 20 µg/ml of RNase A.

The formation of TLS-cTR{alpha}-DNA complexes was studied using a DR4 oligonucleotide HRE (5'-AGGTCAcaggAGGTCA-3') (flanked by HindIII cohesive ends), which was labeled to high specific activity with [32P]CTP using Klenow polymerase (81). GST alone or GST-TLS-1–274 (100–200 ng in 6 µl of slurry of GSH-agarose) was rotated for 60 min at 5 C with PBB alone or PBB containing an excess of purified cTR{alpha} prepared as described previously (81). After the beads were washed three times with 1 ml PBB, the agarose beads were rotated for 30 min at 25 C with [32P]DR4 and washed twice with PBB at 5 C, and the pellets were counted for 32P.

Protein Sequencing
Protein bands of interest were cut from gels that had been stained with Coomassie blue-R250. Gel slices were stored at -20 C until about 5–6 µg of protein were collected. Protein bands were then electroeluted from the gel slices in the presence of endoproteinase Glu-C, and peptide fragments were resolved by HPLC and subjected to automated sequence analysis as previously described (82, 83).

Western Blot Analysis
Gels were electroblotted to nitrocellulose membranes using a semidry transfer apparatus, and the membranes were blocked and incubated with primary antibody as previously described (84). Membranes were probed for TLS immunoreactivity using monoclonal antibody IG10 (hybridoma supernatant, 1:10) directed against the C terminus of human TLS (32). Immunoreactive bands were detected using rabbit anti-mouse IgG-peroxidase and enhanced chemiluminescence (Amersham, Arlington Heights, IL). Expression of GST or GST-cTR{alpha}-1–408 in mammalian cells using pEBG vectors was assessed by probing blots with anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Cell Culture and Transfection
HeLa cells were cultured and transfected by electroporation (41, 50, 81) with 5 µg of pMC110, a GAL4 reporter gene expressing chloramphenicol acetyltransferase (CAT) alone or with the following expression vectors as listed in Table 1Go; GAL4-DBD, GAL4-TLS-N, cTR{alpha}(1–408), cTR{alpha}(120–408), or cTR{alpha}(1–221)VP16. After incubation for 48 h with or without T3, cells were harvested for assay of CAT activity (41, 50, 81). The amount of protein used in the assays was adjusted to keep the percent conversion of [14C]chloramphenicol below 40%, which is in the linear range. CAT activity values were normalized to represent the percentage of [14C]chloramphenicol acetylated by a specific amount of cell protein in 16 h at 37 C. All experiments were performed using duplicate or triplicate flasks and were repeated at least three times. Variation among duplicate or triplicate flasks was less than 10%. Human 293T cells were transfected with pEBG or pEBG-cTR{alpha}-1–408 by CaPO4 coprecipitation as previously described (48). Forty hours later, half of the flasks received 1 µM T3 for 1 h, and the cells were then harvested for the preparation of cytosol and nuclear extracts by the method of Dignam et al. (49) as described earlier. Equal amounts of cell protein (~1 mg) were incubated with GSH-agarose for 1 h at 4 C, and the GSH-agarose bound proteins were then analyzed by Western blotting as described above.


    ACKNOWLEDGMENTS
 
We thank Paul van der Saag for GST-mRXR{alpha}, Peter Kushner and Gabriela Lopez for GST-hER-1–185 and 1–282, Myles Brown for GST-hER-278–595, Leonard Freedman for rGR-440–525, and Bruce Mayer for pEBG. We thank Ron Beavis at the Seaver Mass Spectrometry and Protein Chemistry Laboratory at the Skirball Institute of Biomolecular Medicine at the New York University Medical Center (NYUMC) for protein sequencing.


    FOOTNOTES
 
Address requests for reprints to: H. H. Samuels, Department of Medicine and Pharmacology, TH-454, New York University Medical Center, 550 First Avenue, New York, New York 10016. e-mail: samueh01@mcrcr.med.nyu.edu

This research was supported by NIH Grant DK-16636 to H.H.S. and a Senior Fellowship Award DK-09211 to C.A.P. D.R. is supported by NIH Grant CA-60945 and is a Pew Scholar in Biomedical Sciences and a Stephen Birnbaum Scholar of the Leukemia Society of America. Oligonucleotide synthesis was provided by the NYUMC General Clinical Research Center (NIH, NCRR, Grant M01RR00096). H.H.S. and D.R. are members of the NYUMC Cancer Center (Grant CA-16087). Sequence analysis and database searches were through the NYUMC Research Computing Resource which received support from the National Science Foundation (Grant DIR-8908095).

1 On sabbatical leave from the Department of Pharmacology, New York Medical College, Valhalla, NY. Back

Received for publication September 4, 1997. Accepted for publication October 3, 1997.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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