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Home , Retinoic acid receptor, Thyroid hormone receptor

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9525-9529, October 1995

A nuclear hormone -associated that inhibits transactivation by the hormone and receptors (/steroid receptor/two-hybrid system) THOMAS P. BURRIS*, ZAFAR NAWAZ, MING-JER TSAI, AND BERT W. O'MALLEYt Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030 Contributed by Bert W. O'Malley, July 7, 1995

ABSTRACT Nuclear hormone receptors are transcrip- system, we attempted to identify the that interacts tion factors that require multiple protein-protein interactions with this region of TR. In the course of these experiments, we to regulate the expression oftheir target . Using the yeast serendipitously identified a protein that inhibits the ability of two-hybrid system, we identified a protein, thyroid hormone TR and the RAR to transactivate target genes. receptor uncoupling protein (TRUP), that specifically inter- acts with a region of the human thyroid AND (TR) consisting of the hinge region and the N-terminal por- MATERIALS METHODS tion of the binding domain in a hormone-independent Plasmid Construction. The GAL4 DBD-TR168-259 yeast manner. Interestingly, TRUP inhibits transactivation by TR expression plasmid (pASlcyh-TR168-259) (where DBD is and the but has no effect on the estrogen DNA binding domain) was constructed by inserting the receptor or the in mammalian cells. We BamHI/HindIII (filled) fragment of pAI3Agal TR168-259 also demonstrate that TRUP exerts its action on TR and (13) into the BamHI/Sal I (filled) sites of pAS1-cyh (14). retinoic acid receptor by interfering with their abilities to pTRE2LacZ was constructed by inserting two copies of a interact with their DNA. TRUP represents a type ofregulatory DR4 oligonucleotide (15) into the Bgl II site of PC3 (16). protein that modulates the transcriptional activity of a sub- pl7merLacZ has been described previously (17). To construct class of the nuclear hormone receptor superfamily by pre- pCBUB GAL TR168-456, HindlIl-digested (and filled) pAB venting interaction with their genomic response elements. Gal TR168-456 was partially digested with Bgl II. The result- ing Bgl II-HindIll (filled) fragment was inserted into the Nco Members of the nuclear hormone receptor superfamily are I-Pvu II sites of pCBUB linker (17). YepTR(3 was constructed ligand-activated transcription factors (1-4). This superfamily by first inserting an EcoRI fragment containing the entire includes the receptors for steroid hormones, thyroid hor- coding sequence of human TR f3 (hTR,B) into BCpV2 (18). mones, lipophilic vitamins such as vitamins A and D, and the The Afl II/Kpn I fragment of this vector was then subcloned orphan receptors, which have structure consistent with other into the corresponding sites of the yeast expression vector superfamily members but have no identified ligands. Nuclear YepVl (18). pCMV4-TRUP (where TRUP is TR incoupling receptors regulate expression by interacting with specific protein) was constructed from pBK-CMV-TRUP. pBK-CMV- DNA sequences (response elements) in the promoters of TRUP was created by first inserting the Xho I fragment of target genes (5, 6). Receptors for the classical steroid hor- pACT-TRUP (cor 1.3) into the Xho I site of pBK-CMV mones bind to their respective response elements in a ligand- (Stratagene) followed by sequencing to determine the correct dependent manner; however, some receptors such as the orientation. The HindIII-Xba I fragment ofpBK-CMV-TRUP (TR) and retinoic acid receptor was then inserted into the HindIII-Xba I sites of pCMV4 (19) (RAR) bind to their response elements in a ligand-indepen- to create pCMV4-TRUP. dent manner. The receptors modulate the rate of transcription Two-Hybrid System. The yeast strain y190 (MATa, leu2-3, of target genes by interacting with basal transcription machin- 112, ura3-52, trpl-901, his2-D200, ade2-101, gal4Agall8OA ery either directly (7-10) or indirectly via TATA binding URA3 GAL-lacZ, LYS GAL-HIS3, cyhr) containing pASlcyh protein-associated factors (11). The interaction of nuclear TR168-259 was transformed with a human B-lymphocyte receptors with the basal transcriptional machinery may also be cDNA library in pACT (14) and plated on SC medium lacking regulated by ligand binding. TR is an example of this level of tryptophan, leucine, and histidine (containing 25 mM 3-ami- regulation by ligand binding. In the absence of ligand, the notriazole) as described by Durfee et al. (14). His' colonies ligand binding domain (LBD) of TR interacts with the basal exhibiting ,B-galactosidase activity using the filter lift assay were , TFIIB. In the presence of ligand this further characterized. 3-Galactosidase activity was deter- region of TR fails to interact with TFIIB (8). This complex mined using chlorophenyl red f3-D-galactopyranoside (CPRG) ligand-dependent interaction ofTR with TFIIB may play a role as described (14). To recover the library plasmids, total DNA in the mechanism by which TR silences target gene transcrip- from yeast was isolated and used to transform Escherichia coli tion in the absence of ligand and activates target gene tran- scription in the presence of ligand (8, 12). Abbreviations: TRUP, thyroid hormone receptor uncoupling protein; that TR with a TR, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, Recently, we demonstrated must interact retinoid X receptor; ER, ; EMSA, electrophoretic protein(s), termed a corepressor, to silence gene transcription mobility shift assay; DBD, DNA binding domain; hTR(3, human TR ,(; (13). The corepressor interacts with a small region of TR LBD, ligand binding domain; CAT, chloramphenicol acetyltrans- consisting of the hinge region and the N-terminal portion of ferase; tk, thymidine kinase. the LBD (amino acids 168-259). Using the yeast two-hybrid *Present address: Department of Pediatrics, Division of Genetics, School of Medicine, University of California, 10833 Le Conte Ave- nue, Los Angeles, CA 90095. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of Cell payment. This article must therefore be hereby marked "advertisement" in Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX accordance with 18 U.S.C. §1734 solely to indicate this fact. 77030. 9525 Downloaded by guest on September 26, 2021 9526 Biochemistry: Burris et aL Proc. Natl. Acad. Sci. USA 92 (1995) (HB101). Transformants were identified on minimal me- and reporter plasmids pRSV-TR, pRSV-RAR, pRSV-RXR, dium lacking leucine and containing ampicillin. To ensure pRSV-ER, DR4-tkCAT, DR5-tkCAT, DR1-tkCAT, and that the correct cDNAs were identified, library plasmids ERE2-tkCAT have been described (15, 21-23). isolated were retransformed into y190 containing Electrophoretic Mobility Shift Assay (EMSA). All pASlcyhTR168-259 and 3-galactosidase activity was deter- used in the EMSA were in vitro translated using the TNT mined. The specificity of the interaction of TRUP with TR system (Promega) according to the manufacturer's protocol. was determined by mating y190 containing pACT-TRUP The plasmids used for in vitro of the receptors, with the strain y187 (MATa, gal4, gal80, his3, trpl-901, pT7-TR, pT7-RAR, pT7-RXR, and pT7-ER, have been de- ade2-101, ura3-52, leu2-3,-112, URA3 GAL-lacZ met-) con- scribed (15, 21-23). pBK-CMV-TRUP was used to in vitro taining either pAS1-SNF, pAS1-CDK2, pAS1-, or pAS1- translate TRUP. The EMSA was performed using unlabeled lamin. The /3-galactosidase activity of these diploids was translated receptors that were preincubated for 15 min at room examined using the filter lift and CPRG methods. The temperature with unlabeled in vitro translated TRUP in a total interaction of TRUP with GAL TR 168-456 and full-length volume of 8 ,ul [containing 2 ,ul of 5x bandshift buffer (1x TR was examined using a modified two-hybrid system. The bandshift buffer = 60 mM KCl/5 mM MgCl2/20 mM Hepes, yeast strain BJ2168 (genotype MATa, prcl-407, Prbl-1122, pH 7.5/2 mM dithiothreitol/10% glycerol)]. Approximately pep4-3, leuZ trpl, ura 3-52) was transformed with either equal amounts of receptor and TRUP were incubated, as pCBUB GAL TR 168-456 or YepTR and their correspond- determined by SDS/PAGE analysis of 35S-labeled protein ing reporters (pl7mer LacZ and pTRE2 LacZ, respectively). made in parallel with the unlabeled protein used in the EMSA. Also included was empty pACT for controls or pACT- Two microliters of a solution containing 0.1 ng of 32P-labeled TRUP. ,B-Galactosidase activity was quantitated as de- DNA response element and 300 ng of pGEM (digested with scribed (17). Hpa II) was added to the reaction mixture and incubated an Transfections. Transfections of the Lmtk- cells and chlor- additional 15 min at room temperature. The response elements amphenicol acetyltransferase (CAT) assays were performed as (DR1, DR4, DR5, and estrogen receptor response element) described (20). Equivalent amounts ofempty expression vector and the methods for their labeling have been described (15, (pCMV4) were included in cells cotransfected with only a 23). Protein-DNA complexes were resolved on a 5% poly- receptor and reporter and not pCMV4-TRUP. The expression acrylamide gel (15).

C D E a B 1456-- hTRP LI qp-lw-v-lv, DBD t2 T3 LBD 1 68 259 GAL TR 168-259 GAL4 DBD 168 456 GAL TR 168-456 GAL4 DBD 1 266 I'l s X\eeESSS/ 1 .1 I GAL-ACT TRUP _ , s . \@ \P \, E- \J s2 GAL ACT TRUP b 1 MP1GKKAKGKKV,A P A P A V V K 20 21 KQEAKKVVNPLF E R P K N F G 40 41 I G QD I Q P KRD L T 3R F V K W P R Y 60 61 I R L Q R Q R A I L Y K IR L K V P P A I 80 81 N Q F T Q A LD R Q T A'T QL L K L A H 100 101 KY R P ET K Q EK K Q R L L A R A E K 120 121 P P V L R A G V 140 KAAGKGDVPTKR: a 141 NTVTTLVENKKA FIG. 1. Identification of protein that 161 V D P I E L VV F L P A:L C R M GV P 180 interacts with the hTR,3 using the yeast 181 YCIIKGKARLGR:L V HR K T C T 200 two-hybrid system. (a) Schematic of full- 201 T V A FT Q V N S E D K (GALAKLVE 220 length TR, the GAL4 DBD TR (amino 221 A I R T N Y NDRYDE.I R R H W G G N 240 acids 168-259) fusion protein used for 241 V L G P K S V A R I A K: 261 L A T K L G screening a cDNA library fused to the GAL4 activation domain (GAL-ACIT), the GAL4 DBD-TR (amino acids 168- C 120' 30 456) fusion protein, and the GAL4-ACT fusion protein identified, which specifi- ; 100 cally interacts with the 168- to 259-amino acid fragment of TR. T2, T3, and r4 rep- J 80 ;t e20 _ resent previously characterized transacti- 'u vation domains (6). (b) The identified 60' -I cDNA encodes a previously identified -0;A Ui 0 10 protein, surf-3 40- j 3 __ ~~~~~ribosomal266-amino proteinacid (GenBank accessionor L7a li, no. M36072), which is in frame with the 20 N-terminal GAL4-ACT. After functional

characterization, we termed the identified 0-_ O - __ protein TRUP. (c) TRUP interacts with GAL - TR 168-456 + + TR,B the entire LBD of TR; however, it de- creases the transcriptional activity of full- GAL-ACT-TRUP + GAL-ACT.-TRUP _ + length TR. Downloaded by guest on September 26, 2021 Biochemistry: Burris et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9527 RESULTS AND DISCUSSION acids 168-456) of TR to the GAL4 DBD (GAL TR 168-456) (Fig. la) and examined the ability ofTRUP to interact with this To identify proteins that interact with and mediate and/or protein in the two-hybrid system. As shown in Fig. lc, coex- modulate the activity of TR we employed the yeast two-hybrid pression of GAL-ACT TRUP increased the transcriptional system (24). A fragment of TR consisting of the hinge region activity of GAL TR 168-456. More importantly, we examined and the N-terminal portion of the LBD (amino acids 168-259) the ability of TRUP to interact with full-length TR utilizing its was fused to the GAL4 DBD (Fig. la). Previously, we dem- own DBD and not the GAL4 DBD. Intriguingly, coexpression onstrated that this region of TR interacts with proteins that are of GAL-ACT TRUP significantly decreased the transcrip- required for its function (13). The GAL4 DBD-TR 168-259 tional activity of full-length TR (Fig. lc Right). construct was used to screen a human B-lymphocyte cDNA To investigate the inhibitory activity of TRUP on the library (fused to the GAL4 ) (14). function of TR, we subcloned the TRUP coding sequence Approximately 500,000 recombinants were screened, of which (without the GAL4 activation domain) into a mammalian 20 were classified as positive. One positive clone, cor 1.3, expression vector. Lmtk- cells were cotransfected with an interacted with TR but not with other unrelated proteins such expression vector for the full-length TR and a reporter con- as lamin, p53, cyclin, and SNF (data not shown). cor 1.3 cDNA taining a TR response element (DR4) upstream of the thy- (-900 bp) was recovered from the yeast, sequenced, and midine kinase promoter-CAT gene reporter (tkCAT) (15) in shown to contain an open reading frame (ORF) of 798 bp (266 the presence or absence of an expression vector for TRUP. In amino acids) (Fig. 1 a and b). The initiation codon in the cells transfected with TR and the reporter, normal hormone- identified ORF was in frame with the sequence of the GAL4 dependent activation of the reporter was observed (Fig. 2a, activation domain. Based on its interaction with TR and lanes 3 and 4). However, in cells cotransfected with TRUP, TR subsequent functional characterization, we named the protein was no longer able to activate reporter gene transcription (Fig. encoded by cor 1.3 TRUP. Sequence comparison indicated 2a, lane 6). The inhibitory effect of TRUP on TR transacti- that TRUP is identical to the previously identified surf-3, vation was dose dependent (data not shown). Furthermore, in PLA-X, and the L7a ribosomal protein cDNAs (25-27). Al- the presence of TRUP, TR was less effective in silencing the though this protein has been recovered previously from ribo- basal transcription of a reporter gene in the absence of ligand somal preparations, and has been shown to be contained (data not shown). within the nucleus/nucleolus by histochemistry, it has no To examine whether the inhibitory effect of TRUP is proven function (26). specific for TR, we also examined the effects of TRUP on To initially characterize the interaction of TRUP with the RAR, a nuclear hormone receptor closely related to TR. intact LBD of TR, we fused the hinge and entire LBD (amino Lmtk- cells were cotransfected with an expression vector for a b

-T R- - X + l + + _ RAR - t J1+±jii T3TRUPI --T-.-l+.-+I t - - +

0.

1 2 3 4 5 6 1 2 3 4 5 6 C d IER1--+++ 1 TRUP _ ,,51: _:~ TRUP _ | +

...E' -.'ML do.fr. s.e ::.:.:: "i -t-S., ...

4 5 1 2 3 4 5 6 1 2 3 4 5 6 FIG. 2. Inhibition of TR- and RAR-mediated transactivation by TRUP in mammalian cells. (a) Representative data from CAT assays from Lmtk- cells transiently transfected with pRSV-TR (1 ,ug) and DR4-tkCAT (5 ,ug) in the presence or absence of pCMV4-TRUP (5 jig). (b) pRSV-RAR (1 ,ug) and DR5-tkCAT (5 jig) were cotransfected into Lmtk- cells in the presence or absence of pCMV4-TRUP (5 jig). (c) CAT assay data from cells transfected with pRSV-ER (1 jig) and ERE2-tkCAT (5 jig) in the presence or absence of pCMV4-TRUP (5 ,g). (d) pRSV-RXR (1 gg) and DR1-tkCAT (5 jig) were cotransfected into Lmtk- cells in the presence or absence of pCMV4-TRUP (5 jig). T3, addition of 1 ,uM ; RA, addition of 1 ,uM all-trans-retinoic acid; 9-cis, addition of 1 ,tM 9-cis-retinoic acid; E2, addition of 1 /LM estradiol. RXR, retinoid X receptor; ER, estrogen receptor. Downloaded by guest on September 26, 2021 9528 Biochemistry: Burris et al. Proc. Natl. Acad. Sci. USA 92 (1995) full-length RAR and a reporter containing a RAR response (Fig. 2 c and d). Overexpression of TRUP alone did not affect element (DR5) upstream of tkCAT (15) in the presence or any of the reporters examined. absence of an expression vector for TRUP. In cells transfected We were able to demonstrate in the two-hybrid system that with RAR and the reporter, normal hormone-dependent TRUP interacts with and increases the transcriptional activity activation of the reporter was observed (Fig. 2b, lanes 3 and 4) of GAL4 DBD-TR 168-456; however, TRUP decreases the and, in a manner similar to TR, overexpression of TRUP transcriptional activity of full-length TR. The major difference antagonized the ability of RAR to transactivate (Fig. 2b, lane between these two TR constructs is that the GAL4 DBD-TR 6). To examine the specificity of the inhibitory effects of 168-456 utilizes the GAL4 DBD and the full-length TR TRUP, we overexpressed TRUP along with ER and RXR and construct uses its natural DBD. Therefore, we hypothesized their respective reporters. TRUP had no effect on the abilities that TRUP may inhibit full-length TR function by interfering of these nuclear receptors to transactivate their target genes with the natural DNA binding function of TR, while having no b a TR ++|++- RXR + ++ + -_ TRUP--X + + - T3 X- --lL

TR-RXR_ N ii :. IA'bmw'kl. _X5 n S ^^ t-oog4NS RAR-RXR-fr .NS NS_- NS_- ..- -'.4.Pi'.z" :;i. .1 .:i::a i+

i. ::,..'

1 2 34 56 1 2 3 4 5 6 C .ER + + |+ + --

TRUP. - .-I + + - + d E2 |_|+ 1-1 + 1 = RXR +__+ TRUP - - + + _ + ER-*-` S*w 9-cis RA - + - + -

RXR-*

1 2 3 4 5 6 1 2 3 4 5 6 FIG. 3. TRUP prevents transactivation by TR and RAR by inhibiting their abilities to interact with DNA. (a) EMSA demonstrating the ability of TRUP to inhibit TRPRXR heterodimers binding to a 32P-labeled DR4 element. (b) TRUP inhibits the ability of RAR-RXR heterodimers to bind to a DR5 element in an EMSA. (c) ER's ability to interact with an estrogen receptor response element is not affected by the presence ofTRUP. (d) RXR's ability to bind to a DR1 element is also unaffected by the presence of TRUP. T3, addition of 1 ,uM triiodothyronine; RA, addition of 1 iMM all-trans-retinoic acid; 9-cis, addition of 1 ,uM 9-cis-retinoic acid; E2, addition of 1 MkM estradiol; NS, nonspecific complex that is present in lysate controls. Downloaded by guest on September 26, 2021 Biochemistry: Burris et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9529

effect on the heterologous binding domain of the GAL4 appears to contribute to the highly controlled and graded DBD-TR 168-456 chimera. We addressed this possibility by functional response that results from ligand activation of examining the ability of in vitro translated TR, RAR, ER, and receptors in animal cells. RXR to bind to their respective response elements in the presence and absence of in vitro translated TRUP in an EMSA. We thank members of the laboratory for critically reading the An equal amount of RXR was added to TR and RAR in the manuscript. We also thank Stephen Elledge for providing the yeast EMSA to facilitate the detection of retarded complexes. As two-hybrid system and lymphocyte library. This work was supported by shown in Fig. 3a, TRRXR heterodimer bound to a 32P-labeled a grant from the American Cancer Society. DR4 element can be detected in the presence and absence of 1. Evans, R. M. (1988) Science 240, 889-895. hormone (lanes 1 and 2). However, if the heterodimer is 2. Beato, M. (1991) FASEB J. 5, 2044-2051. preincubated with an equal amount of TRUP (as determined 3. Green, S. & Chambon, P. (1988) Trends Genet. 4, 309-314. by SDS/PAGE analysis) the ability of TR-RXR to bind the 4. Baniahmad, A., Burris, T. P. & Tsai, M. J. (1994) in Mechanism element is significantly decreased (Fig. 3a, lanes 3 and 4). of Steroid Hormone Regulation of Gene Transcription, eds. Tsai, Similar results were noted with the RARRXR heterodimer M. J. & O'Malley, B. W. (CRC, Boca Raton, FL), pp. 1-24. (Fig. 3b). A stoichiometric amount of TRUP is able to inhibit 5. Glass, C. K. (1994) Endocr. Rev. 15, 391-407. the ability of RAR-RXR heterodimers to bind to a DR5 6. Cooney, A. J. & Tsai, S. Y. (1994) in Mechanism of Steroid element (Fig. 3b). The presence of hormone does not affect the Hormone Regulation of Gene Transcription, eds. Tsai, M. J. & ability of TRUP to interfere with DNA binding of TR-RXR or O'Malley, B. W. (CRC, Boca Raton, FL), pp. 25-59. RAR-RXR heterodimers, indicating that the interaction of 7. Ing, N. H., Beekman, J. M., Tsai, S. Y., Tsai, M. J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 17617-17623. TRUP with TR and RAR is hormone independent (Fig. 3 a 8. Baniahmad, A., Ha, I., Reinberg, D., Tsai, S., Tsai, M. J. & and b). Consistent with our transfection data, TRUP does not O'Malley, B. W. (1993) Proc. Natl. Acad. Sci. USA 90,8832-8836. alter the DNA binding activity of either ER (Fig. 3c) or RXR 9. MacDonald, P. N., Sherman, D. R., Dowd, D. R., Jefcoat, S. C., (Fig. 3d). Jr., & DeLisle, R. K. (1995) J. Biol. Chem. 270, 4748-4752. Our results demonstrate that TRUP interacts specifically 10. Blanco, J. C., Wang, I. M., Tsai, S. Y., Tsai, M. J., O'Malley, with a subclass of nuclear hormone receptors and antagonizes B. W., Jurutka, P. W., Haussler, M. R. & Ozato, K. (1995) Proc. their ability to transactivate by inhibiting their binding to Natl. Acad. Sci. USA 92, 1535-1539. specific DNA response elements. Another protein, calreticu- 11. Jacq, X., Brou, C., Lutz, Y., Davidson, I., Chambon, P. & Tora, lin, has been demonstrated recently to have a similar effect on L. (1994) Cell 79, 107-117. the receptor, 12. Casanova, J., Helmer, E., Selmi-Ruby, S., Qi, J. S., Au-Fliegner, several nuclear receptors, including androgen M., Desai-Yagnik, V., Koudinova, N., Yarm, F., Raaka, B. M. & , and RAR (28, 29). in- Samuels, H. H. (1994) Mol. Cell. Biol. 14, 5756-5765. hibits the DNA binding of these receptors by binding directly 13. Baniahmad, A., Leng, X., Burris, T. P., Tsai, S. Y., Tsai, M. J. & to their DBD (28, 29), whereas TRUP likely exerts its effects O'Malley, B. W. (1995) Mol. Cell. Biol. 15, 76-86. by interacting primarily with a region of the receptor that is 14. Durfee, T., Becherer, K, Chen, P. L., Yeh, S. H., Yang, Y., C-terminal of the DBD. The precise mechanism by which Kilburn, A. E., Lee, W. H. & Elledge, S. J. (1993) Genes Dev. 7, TRUP inhibits the DNA binding activities of TR and RAR is 555-569. not clear and several possibilities exist. When TRUP binds to 15. Cooney, A. J., Leng, X., Tsai, S. Y., O'Malley, B. W. & Tsai, M. J. the receptor just C-terminal of the DBD, it may either (1993) J. Biol. Chem. 268, 4152-4160. allosterically alter the DBD rendering it inactive or it may 16. Nawaz, Z., Tsai, M. J., McDonnell, D. P. & O'Malley, B. W. with DNA. Alter- (1992) Gene Exp. 2, 39-47. sterically hinder the DBD from interacting 17. Nawaz, Z., Baniahmad, C., Burris, T. P., Stillman, D. J., natively, TRUP may interfere with the dimerization properties O'Malley, B. W. & Tsai, M. J. (1994) Mol. Gen. Genet. 245, of TR and RAR preventing DNA binding. We prefer the 724-733. former model based on the ability of TRUP to inhibit the DNA 18. McDonnell, D. P., Pike, J. W., Drutz, D. J., Butt, T. R. & binding of TR monomers as determined in an EMSA (data not O'Malley, B. W. (1989) Mol. Cell. Biol. 9, 3517-3523. shown). 19. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H. & Russell, The unusual ability of TRUP to regulate the DNA binding D. W. (1989) J. Biol. Chem. 264, 8222-8229. abilities of only a subset of the steroid/thyroid/vitamin recep- 20. Baniahmad, A., Tsai, S. Y., O'Malley, B. W. & Tsai, M. J. (1992) tor superfamily may represent again another level of regula- Proc. Natl. Acad. Sci. USA 89, 10633-10637. tory complexity for the nuclear hormone receptor superfamily 21. Leng, X., Blanco, J., Tsai, S. Y., Ozato, K., O'Malley, B. W. & Tsai, M. J. (1994) J. Biol. Chem. 269, 31436-31442. at the level of the target gene. Ligand binding favors dimer- 22. Klein-Hitpass, L., Tsai, S. Y., Greene, G. L., Clark, J. H., Tsai, ization of these receptors and specific high-affinity interaction M. J. & O'Malley, B. W. (1989) Mol. Cell. Biol. 9, 43-49. with DNA. Negative regulators such as calreticulin can spe- 23. Beekman, J. M., Allan, G. F., Tsai, S. Y., Tsai, M. J. & O'Malley, cifically oppose this interaction, with respect to GR, AR, and B. W. (1993) Mol. Endocrinol. 7, 1266-1274. RAR, while TRUP acts similarly for TR and RAR. Both 24. Fields, S. & Song, 0. (1989) Nature (London) 340, 245-246. negative regulators have been shown to function in transfec- 25. Kozma, S. C., Redmond, S. M., Fu, X. C., Saurer, S. M., Groner, tion experiments in intact cells. Although the physiological B. & Hynes, N. E. (1988) EMBO J. 7, 147-154. relevance is not proven to date, such negative regulators may 26. Giallongo, A., Yon, J. & Fried, M. (1989) Mol. Cell. Biol. 9, represent additional examples of a growing number of cellular 224-231. H. L. & B. and that oppose function, 27. Chazenbalk, G. D., Wadsworth, Rapoport, (1990) factors Mol. Cell. Endocrinol. 68, R25-R30. among which are heat shock proteins, phosphatases, corepres- 28. Bums, K, Duggan, B., Atkinson, E. A., Famulski, K. S., Nemer, sors, and chromosomal histone structure. Variation in con- M., Bleackley, R. C. & Michalak, M. (1994) Nature (London) 367, centration of such regulators could determine the relative 476-480. hormonal response of different tissues during development or 29. Dedhar, S., Rennie, P. S., Shago, M., Hagesteijn, C. Y., Yang, H., in the differentiated state. Although much more complex than Filmus, J., Hawley, R. G., Bruchovsky, N., Cheng, H., Matusik, previously thought, this myriad of positive and negative factors R. J. & Giguere, V. (1994) Nature (London) 367, 480-483. Downloaded by guest on September 26, 2021