Studies on the Mechanism of Functional Cooperativity between ...

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expression. Replacing estrogen with either lo-' M tamoxifen. (Tam) or lo-' M nafoxidine (Naf) blocked induction. Thus,. DNA binding of a transcriptionally inactive ...
THEJOURNAL OF BIOLOGICALCHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 25, Issue of September 5, pp. 166&1-16690,1991 Printed in U.S.A.

Studies on the Mechanism of Functional Cooperativity between Progesterone and Estrogen Receptors* (Received for publication, April 17, 1991)

M. Suzanne BradshawS, Sophia Y. Tsai, Xiaohua Leng, Alan D. W. Dobsons, Orla M. Conneely, Bert W. O’Malley, and Ming-JerTsaill From the Department of Cell Biology,Baylor College of Medicine, Houston, Texas 77030

Steroidresponseelements(SREs)cooperate with copies or tightly clustered with other cis-acting DNA elements many different cis-acting elements including NF- 1 or SREs (1, 3, 8-13). The tryptophan oxygenase gene (8), sites, CACCC boxes, and other SREs to induce target murine mammary tumor viruslong terminal repeat (ll), and gene expression (Schule, R., Muller, M., Otsuka-Mu- vitellogenin A2 and B1 genes (9) contain multiple SREs. In rakami, H., and Renkawitz, R. (1988) Nature 332, addition, the murine mammary tumor virus long terminal 87-90; Strahle, U., Schmid, W., and Schutz, G . (1988) repeat (11)and the rat tryptophan oxygenase gene (1)contain EMBO J. 7,3389-3395). Induction of gene expression other cis-acting elements in close conjunction with SREs. In can be additiveor synergistic with respect to the levelall of these cases, when one of the SREs or the adjacentcisof activation by either transactivators. Two mecha- acting elements are mutated, steroid responsiveness is greatly nisms have been proposed for how synergism occurs: diminished. Thus, SREs interact synergistically witheach 1)cooperative binding of transcriptional activators to otherandwithother cis-acting elementsto induce gene DNA or 2) simultaneous interaction of individually expression. The mechanism of synergism between SREs and bound activators with a common target protein. We have shownpreviously that cooperativebinding of various cis-acting elementsis not known. receptors is important for synergism between two pro- Ptashne (14) has proposed two models by which synergistic gesterone response elements (PREs). Here we showed activation occurs. First, synergistic activation canoccur when that an estrogen response element (ERE) and a PRE two factors bindcooperatively to adjacentcis-acting elements. can also functionally cooperateand this synergismbe- Second, synergismcan occur when twofactors bind independtween an ERE and a PRE is not contributed by coop- ently to DNA but simultaneously interact with a third tranerative DNA binding. Furthermore, we have demon- scription factor to initiate transcription. Tsai et al. (15) and strated that the activation domains of the progesterone Klein-Hitpass et al. (7) have demonstrated that progesterone receptor (PR) (ClAct) are required for synergismbe- receptor can bind cooperatively to trahscriptionally activate (PREs). Similarly, tween two PREs and sufficient for confirming coop- two progesterone response elements erative binding. However these two activation domains Schmid et al. (16) have demonstratedcooperative binding of an ERE the glucocorticoid receptor to tandem GRE/PREs. Nevertheof PR are not sufficient for synergism between and a PRE. Additional regions within the NH2-termi- less, cooperative binding may not account for the total level nal andCOOH-terminal domains are also required for of synergism observed between SREs. Carey et al. (17) and synergistic interaction between two heterologous Lin et al. (18) have demonstrated recently that synergism SREs. occurs between GAL4 binding sites and between GAL4 and ATF sites even when all sites are saturated with activators. This result is consistent with the hypothesis that activators cooperate by simultaneously touching a target protein. The Steroid hormones control gene expression via specific in- observation that ER can squelch the activation of a progesteractions of their cognate receptors with steroid response terone-responsive target gene by the progesterone receptor elements (SREs)’ in the 5”flanking DNA of steroid regulated supports this possibility (19, 20). genes (3-5). Once bound, the mechanism by which receptors In this paper, we demonstrate that the estrogen receptor activate transcription is only partially understood (6,7).Many cooperates functionally with the progesterone receptor, but eucaryotic genes are under the controlof multiple hormones that the synergism does not result from cooperative binding and steroid response elements are usually found in multiple to their respective SREs. The two activation domains of the progesterone receptor areshown to be essential for synergism * This work is supported by grants from the National Institutes of both betweentwo PREsand between an ERE (estrogen Health (to M. J. T. and B. W.O.). The costs of publication of this response element) and a PRE. These two activation domains article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- are sufficientfor partial synergism of two PREs but not sufficient for synergism between an ERE and a PRE. Addiance with 18 U.S.C. Section 1734 solely to indicate this fact. COOH$ Present address: Dept. of Biology, Yale University, New Haven, tional domainsresiding within the NH2-terminal and terminal regions of the heterologous receptors are required. CT 06511. § Present address: Dept. of Microbiology, University College Cork, These data are consistent with the hypothesis progesterthat Cork, Ireland. one andestrogen receptors regulate target genes cooperatively ll To whom correspondence should be addressed. Tel.: 713-798- via simultaneous protein-protein contacts with a target pro6253; Fax: 713-790-1275. tein(s) in the transcriptional machinery. The abbreviations used are: SREs, steroidresponseelements; PREs, progesterone response elements;GRE, glucocorticoid response element; ER, estrogen receptor; ERE, estrogen response element;PR, progesterone receptor; aa, amino acids; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase.

MATERIALS AND METHODS

Construction of Plasmids-Synthetic oligonucleotides containing the tyrosine aminotransferase PRE and the vitellogenin ERE were

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Functional Cooperativity between Progesterone and Estrogen Receptors

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Exprrssion nnd I'nrlinl /'uri/icntion o/ ('lAc!-('lAct protein W;I~ inserted 5' to the TATA hox in the HglII site of pOVCAT-50 (21). expressed as a fusion proteinhy attachment to the 26-klh glutnthionr T h e PRE contained the sequence 5"TGTACACGATGTTCT$-transferase of Srhistosomn jnponicum antl therehy can be p u r i l k l AGCTAC-3'. The ERE contained the sequence 5'-TAGACGTCAunder nondenaturing conditions ( 4 4 ) . p(;RS-2"I"('tArt was tranqHglll ends. I'RECACTGACCTACG-3'.BothhadHnmHIand formedinto k;. e d i strain JM109. Fusionprotein (;ST-('IArt wab TKCAT#S is desrrihed in Tsai r f nl. (15). preparedfrom /+,'. roli cell extract hy affinitychromatoCraphyon Expression vectors for production of wild-t.ype steroid hormone receptors were: 1) pS9K (22, 23) containing the chicken progesterone prepacked glrltathione-Sepharose 4 H (Pharmaria). ('sing procedures furnished hv I'harmacia we prlrifietl the fusion protein except thnt receptor cnNA and 2 ) AHER/p91023 (21) containing the human the fusion protein was eluted with 1 0 m u glrltathione ISipma) in TrO estrogenreceptorcDNA.Oligonucleotidesite-directedmutants of mM Tris-HCI and 15"; glycerolelutionhuffer. The glutathione S'progesterone receptor (1'13) were constructed as descrihed in Dohson transferase carrier was removed from fusion protein by incuhation r f n/. (24). I'IICIH a n d I'RCICZ are deletion mutants desrrihed in More than 95'; cleavage Carson r f 01. ( 2 5 ) . Receptor recomhinants containing activation do- with thromhin (Hoehringer Xlannheim)(38). occurred after 2-h incubation at room temperature and an enzymrmain were constructed with a 275-hase pair Sacl-/findlll (aa 281368) fragment o f the progesterone receptor rDNA that contains only t o - s u h s t r n t e m t i o o f 1 : 1 0 0 . ~ ~ l r ~ t a t h i o n e w a s t h e n r e m r ~ v r r l a s d e s c r i l ~ r d hy Smith and .Johnson ( 4 4 ) . (;lutnthioneS'-transfemseandothrr the DNA hinding domain. An A'K start site and stop codon were added hv oligonucleotide insertions. Activation domains were inserted glutathione binding proteins were partially rrmovedby affinity rhromatography. ClAct preparation was then drsalted and concmtrntetl at the Hindlll sties. pC1-5 contains a 160-nucleotide Hindlll-Hnnll hv using Centricon-10 concentrater antl storedat -70 "c'. lhntl-shift fragment (na 868-421 ) . T h e I h l l site was hlrlnt-ended with1'4 DNA polvmerase antl Hindlll linkers were added. pC1-I8 contains 245- analyses of C 1Act on two I'IiEa probes were carried o u t as clr.;cril)rtl previously (15). nucleotide HnnlI fragment (at1 199-281) hlunt-ended with T4 DNA polymerase and Hintllll likers added. Both recombinants were inserted in the /