Chimeric Receptors Used to Probe the DNA-Binding Domain of the Estrogen and Glucocorticoid Receptors1

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(CANCER RESEARCH (SUPPL.) 49, 2282s-2285s, April IS, 1989) Chimeric Receptors Used to Probe the DNA-binding Domain of the Estrogen and Glucocorticoid Receptors1

Stephen Green2 and Pierre ChambÃ³n3 Laboratoire de GÃ©nÃ©tiqueMolÃ©culairedesEucaryotes du CNRS, UnitÃ©184de Biologie MolÃ©culaireetde GÃ©nieGÃ©nÃ©tiquedel'INSERM, Institut de Chimie Biologique, FacultÃ©deMÃ©decine,II rue Humann, 67085 Strasbourg, France

Abstract then the exchange of this region of the hER with that of the hGR should result in a chimeric receptor able to activate Steroid hormone receptors activate specific gene transcription by transcription of glucocorticoid-responsive genes. To facilitate binding as hormone-receptor complexes to short DNA enhancer-like the exchange of region C between the two receptors, unique elements termed hormone response elements. The DNA-binding domain restriction enzyme recognition sites were created at the 5' (termed region C) is a highly conserved 66-amino acid region that contains two subregions (CI and CII) analogous to the "zinc fingers" of transcrip (Kpnl) and 3' (Xho\) ends of this region in the complementary tion factor 111A. Using chimeric estrogen receptors, we show that this DNAs of both the hER and hGR (see Fig. l, HE28 and region, and in particular the MI j-terminal zinc finger, defines the target GR.CAS). This allows the DNA encoding the cysteine-rich gene specificity of the receptor. We suggest that receptor recognition of region to be exchanged as a cysteine cassette without disturbing the hormone response element is analogous to that of the helix-turn- the reading frame. The insertions of the Kpnl and Xhol sites helix DNA-binding motif in that the receptor binds to DNA as a dimer introduce, respectively, two (glycine and threonine) and three with the first zinc finger lying in the major groove recognizing one-half (alanine, arginine, and glutamic acid) new amino acids (Fig. 1). of the palindromic hormone response element and protein-DNA interac In the presence of estradici, HE28 stimulated vit-tk-CAT but tion is stabilized through nonspecific DNA binding and dimer interactions not MMTV-CAT (data not shown). No stimulation was seen contributed by the second zinc finger. when using the parent expression vector, pSGl, which does not contain hER sequences (Fig. 4B, Lane 2). Introduction We next exchanged the cysteine cassette of the hER with that of the hGR, producing the ER-GR.CAS chimera (Fig. 1). Steroid hormone receptors are members of a large family of ligand-activated transcription enhancer factors (1,2). Compar Although the ER-GR.CAS did not activate the vit-tk-CAT (Fig. 4B, Lane 6) it did activate MMTV-CAT but only in the presence ison of amino acid sequences combined with functional analysis of in v/iro-generated mutants has defined regions essential for of estradici (Fig. 4B, Lane 16). This indicates that the target the three major functions of the "nuclear receptors," namely gene specificity of the receptor had been changed by replacing ligand binding, DNA binding, and transcription activation. the 66-amino acid core of region C. Based upon amino acid sequence homology (3), the ER4 can be The Two Zinc Finger Regions CI and CII May Correspond to divided into 6 regions (A to F) of which only regions C and E Subdomains within Region C. In order to determine whether the are highly conserved (100 and 94%, respectively). Region C, precise arrangement of the two subregions of region C (CI and which is rich in cysteine and basic amino acids, contains a 66- CII; see Fig. 2) is important for ER function, additional codons amino acid core which is highly conserved among all members were inserted in region C of HE28 at the site of the intron (between Gly215and His216; see Footnote 5 and Fig. 2). These of the nuclear receptor family and contains two subregions (CI and CII) analogous to the zinc-stabilized DNA-binding fingers ER mutants (HE29, HE31, HE45, HE46, HE53, HE54; see of Xenopus transcription factor IIIA (4). The hydrophobic re Fig. 3) were cotransfected into HeLa cells along with vit-tk- gion E contains not only the ligand-binding domain (5) but also CAT. Stimulation of transcription was dependent upon the addition of estradiol (10~8 M) in each case (data not shown). a ligand-dependent transcription-activating region (6). We describe here chimeric receptors in which parts of the The insertion of a single serine codon (HE31) or the insertion region C core of the ER have been replaced with the homolo of a valine and asparagine codon (HE29) produced receptor gous region of the GR. We find that the specificity of target mutants which were at least as active as either the wild type ER gene activation is determined by region C and more specifically (HEO) or HE28 (Fig. 3). The insertion of further codons be by the NH2-terminal putative "zinc finger." tween the valine and asparagine codons of HE29 resulted in a 20 to 60% reduction in transcriptional activity of the receptor mutants [HE45, HE46, HE53, HE54 (Fig. 3)], although notably Results none were inactive. In general, the greater the number of codons Region C Determines Target Gene Specificity. The wild type inserted between regions CI and CII the lower the activity of ER expression vector (HEO) activates transcription (7) of the the ER mutant. Interestingly, mutants containing an insertion ERE-containing vit-tk-CAT (8) reporter gene (Fig. 4B, Lane 4) of codons for proline and glycine (HE53 and HE54), which are but not the GRE of the MMTV long terminal repeat present likely to greatly disrupt protein tertiary structure, still retained in the MMTV-CAT (9) reporter gene (Fig. 4B, Lane 14). We almost wild type activity (80 and 62%, respectively). reasoned that if region C is responsible for the specific inter Region CI Specifies Target Gene Recognition. To further action between the receptor and the ERE of the target gene, evaluate the functional importance of regions CI and CII in the 1Presented at the Symposium on "Glucocorticoid Receptors: Evolution, Struc recognition of the responsive element, we created chimeric ture, Function and Abnormalities," July 14 and IS, 1988, Osaka, Japan. receptors between the ER and GR by replacing either region 2 Present address: Imperial Chemical Industries, Central Toxicology Labs, CI or region CII of the ER with that of the GR. The mutant Alderley, Park, Macclesfield, Cheshire, SK10 4TJ United Kingdom. 3To whom requests for reprints should be addressed. HE31 (Fig. 4A) and the ER-GR.CAS3 (Fig. 4A) were used as 4 The abbreviations used are: ER, estrogen receptor; GR, glucocorticoid recep the parent plasmids for these experiments. Both of these mu- tor; ERE, estrogen-responsive element; GRE, glucocorticoid-responsive element; MMTV, mouse mammary tumor virus; vit, vitellogenin; hER, human estrogen 5M. Ponglikitmongkol, S. Green, and P. ChambÃ³n. Genomic organisation of receptor; hGR, human glucocorticoid receptor; CAS, cysteine cassette. the human oestrogen receptor gene, EMBO J., 7: 3385-3388, 1988. 2282s

transcription, we constructed reporter genes which had syn 1 5SERER-GAS185185 250 302 552 thetic oligonucleotides containing either a consensus ERE or HEO GRE (10) sequence upstream of the tk promoter in the CAT reporter plasmid pBLCAT+ (8). These reporter genes were denoted ERE-tk-CAT and GRE-tk-CAT, respectively. Results 250 3C 58E2 552 1 IE22 obtained using these reporter genes and the same receptor HE28 expression vectors as above indicated that the target gene TAC-GGI-ACC-TGT AtG-GU-CGA-GAA-AtG specificity seen with vit-tk-CAT and MMTV-CAT was main I251AACASN tained (Fig. 4C). Notably, however, HE61 (Lane 4) stimulated 184CTCLEU "TSoT1GR-CAS42125Â° ERE-tk-CAT activity to a level which was about 80% that seen with HEO (Lane 2), and HE62 (Lane 10) stimulated GRE-tk- 486> CAT activity to a level which was about 80% that seen with |GGI-ACC-TGCÂ¡EM ER-GR.CAS (Lane 8). This increased activity of HE61 and HE62 relative to HEO and ER-GR.CAS when using these ATG-GCT-CGA-GAAGLT reporter genes, rather than vit-tk-CAT and MMTV-CAT, may GLUKpnlTHR CYS MET ALA ARG '42'XholGR-CAS185 "86 ' 420'-KÃ•rT"185 487552E2GR-CAS595ER-GR-CAS reflect the close proximity of the responsive elements to the tk promoter. 250302Ã‰Ã‰ IMl Discussion

Fig. 1. Construction of the chimeric receptor ER-GR.CAS. The structures of We show that the highly conserved core of region C is HEO, HE28. GR.CAS, and ER-GR.CAS are shown schematically: the numbers responsible for defining target gene activation and that it is the refer to amino acid positions. Top, division of wild type hER (HEO) into regions first finger which plays the major role in determining this A to F; shaded areas, DNA binding-domain within region C of the human ER and GR. The Kpnl and Xhol sites, created within region C of the HER and hGR specificity. It is possible that the lower activity of the finger to allow the exchange of this region as a cassette (ER.CAS and GR.CAS), are chimeras (HE61, HE62) when compared with either the wild indicated with the additional amino acids introduced into the HE28 protein. For type receptors of those chimeras in which both fingers have further details see Ref. 13. been swapped (ER-GR.CAS) may reflect a distortion of the DNA-protein interaction due to steric hindrance between the -CAG two nonhomologous finger domains (CI and CII). The purified hER DNA binding domain produced in bacteria binds specifi- YHS â€¢¿NEH 1 YA s-0 Gâ€¢Y Nilc1 MUTANT STRUCTURE SEQUENCE ACTIVITY !â€”¿'Q-*T rnvD A. â€”¿WÂ«N IBS 216 250 CC0 Sâ€¢¿C wild type \/Ã•FÃ•1N HEO 100% CV L.Inlron \/ \/E-JY [g]* *TA Ã‡? Inlron

\1rrliÃ¤HlSIQGHNDYM V * HE28 113% W M[K]GGl[!|R)Dgp]GG cC(K)A1 CYEVL-LJ...â€¢¿216 f. . Â« 'lG l â€¢¿â€¢¿ Gly Thr Ala Arg Glu 180 185 l 205,H 221 245 262 215 216 125% non-specific DNA binding 1 HE31 GÂ¡5}i (dimerizalion ?) Gly Thr Ser Ala Arg Glu Fig. 2. Hypothetical structure of hER region C (amino acids 180 to 262). The core of region C (amino acids 185 to 250), which is highly conserved between the different nuclear receptors, is shown as two subregions. CI (amino acids 185 to HE29 123% 215) and CII (amino acids 216 to 250). Each subregion contains four highly conserved cysteine residues which may tetrahedrally coordinate zinc to form a Gty Thr Val Asn Ala Arg Glu zinc finger. Regions CI and CII are separated by an intron located within the codon for Gly2". Another intron is located within the codon for Gly2*4.Amino 215 216 HE45 OfÃ¯TNÃ¯l 50% acids which are highly conserved between the nuclear receptor (more than 7 of 8 sequences) are shown in relief. Basic amino acids are boxed. Those amino acids Gly Thr Val Leu Glu Asn Ala Arg Glu which differ between the hER and hGR in the 66 amino acid core region are marked with an asterisk. HEÂ«

Gly Thr Val Leu Gly Ser Glu Asn Ala Ang Glu tants contain a unique BamHl site (resulting in the insertion of a serine codon) located at the site of the intron in region C. HE53 80% Using HE31 and either regions CI or CII of ER-GR.CAS3, Gly Thr Val Pro Gly Asn Ala Arg Glu chimeric receptors were constructed containing either region

CI of the ER and region CII of the GR (Fig. 4/1, HE61) or HE54 62% region CI of the GR and region CII of the ER (Fig. 4A, HE62). Gly Thr Val Pro Gly Pro Gly Asn Ala Arg Glu Interestingly, when using the finger chimeras, HE61 and HE62, Fig. 3. Transcriptional activity of hER mutants containing insertions between vit-tk-CAT was stimulated by HE61 (Lane 8) but not by HE62 regions Cl and CII. The region of the hER-containing subregions CI and CII is (Lane 10), and the converse was true for MMTV-CAT (see shown schematically (hatched areas) for the wild type ER (HEO) and the hER mutants. Inserted amino acids are indicated. The exact position of the inserted Lanes 18 and 20). Stimulation was estradiol dependent in each amino acids (boxed) is also shown (SEQUENCE) with the numbers referring to case with HE61 having 29% the activity seen with HEO and the position of the wild type amino acids in HEO. HeLa cells were cotransfected HE62 having 18% the activity seen with ER-GR.CAS. with 1 fig of receptor expression vector, 1 >igof vit-tk-CAT reported gene, and 3 fig of internal control plasmid pCHI 10 (Pharmacia). Right, typical value for the In order to demonstrate that these receptor mutants were activity Â±10%)of each of the receptor mutants with respect to HEO and after acting through their cognate-responsive elements to stimulate adjusting for the internal control. 2283s

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Tyr Gly Thr Cys Gly SerHls- Met Ala Arg Glu Met 1841 1(421) (451) 216 (486)| | 251 Kpnl l_â€”_J Xhol BamHI LJL 123456789 10 Fig. 4. A, Schematic organization of the chimeric finger receptors. The structure for region C of the chimeric (HE6I, HE62) and parent (HE31, ER-GR.CAS, ER-GR.CAS3) receptors is shown. The inserted amino acids and new restriction endonuclease sites are indicated. Numbers refer to the position of the amino acids in the wild type ER or, in parentheses, GR. B, transcriptional activity of chimeric finger receptors using reporter genes which contain the 5' flanking regions of steroid hormone-responsive genes. HeLa cells were cotransfected with 1 /ig of receptor expression vector, 3 /ig of internal control plasmili, and l /ig of either the estrogen-responsive (vit-tk-CAT) or glucocorticoid-responsive (MMTV-CAT) reporter genes. Estradiol (Â£2,10~8M) was added where indicated (+). C, transcriptional activity of chimeric finger receptors using reporter genes which contain ER- or GR-binding sites upstream of the tk promoter. HeLa cells were cotransfected as for B using l /ig of receptor gene containing either an estrogen-responsive element (ERE-tk-CAT) or glucocorticoid-responsive element (GRE-tk-CAT). Estradiol (E2, 10~! M) was added in each case (+). cally to DNA in vitro indicating that no other proteins are separate functional domains is suggested by the fact that both involved in the recognition.6 It is possible that region CI deter regions are encoded in separate exons5 and that the insertion mines the specificity of binding by making specific contacts of several amino acids at the position of the intron between CI with some or all of the bases of the responsive element and that and CII reduces, but does not abolish, their ability to activate this binding is stabilized by nonspecific contacts contributed by transcription. It is possible that the amino acids between CI region CII. Notably, region CII differs from region CI in that and CII represent a flexible loop region. Electron microscopic it contains a number of highly conserved basic amino acids (see analysis of receptor-DNA complexes using hER mutants lack Fig. 2) which may be important for contacting the DNA phos phate backbone. Note also that the basic COOH-terminal end ing either region CI or CII indicates that region CI appears to of region C (Met251 to Gly262), which is homologous to the be more important than CII in determining specific DNA constitutive nuclear localization domain of the rat GR (11), and binding (12); a result that correlates well with the findings part of region D (Arg263to Gly271),which also contains a number obtained with the chimeric finger receptors which suggest that of conserved basic amino acids, appear to be important in region CI is important for the specific recognition of the re stabilizing the interaction with DNA.7 sponsive element. Furthermore, an hER mutant [HE27 (13)] in Additional support that regions CI and CII correspond to which two of the cysteine residues which are believed to be important for coordinating zinc in region CI [Cys202and Cys205] 6 S. Mader, L. Tora, S. Green, and P. ChambÃ³n, manuscript in preparation. 1V. Kumar and P. ChambÃ³n, Cell, 55: 145-156, 1988. are replaced with histidines also failed to bind DNA specifically 2284s

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. NUCLEAR RECEPTOR DNA-BINDING DOMAIN when analyzed either by electron microscopy8 or by gel retar should allow these protein-DNA interactions to be further dation. It should be noted, however, that hER mutants in which defined. region CII had been deleted did not bind to DNA in a gel retardation assay (12). Whether this reflects a difference in the Acknowledgments affinity required for binding in the electron microscopic assay compared with the gel retardation assay or is due to the stabi We are grateful to Isabelle Issemann and Elisabeth Scheer for tech lization of the ER-DNA complex by other proteins of the oocyte nical assistance. nuclear extract is unknown. The palindromic nature of hormone-responsive elements sug References gests that the receptor binds as a dimer. Indeed, analysis of the 1. Evans, R. M. The steroid and thyroid hormone receptor superfamily. Science binding of the ER to its responsive element has indicated that (Wash. DC), 240: 889-895, 1988. two receptor monomers bind to a single palindromic ERE and 2. Green, S.. and ChambÃ³n, P. Nuclear receptors enhance our understanding has suggested that dimerization results from interaction be of transcriptional regulation. Trends in Genetics, 4: 309-314, 1988. 3. Krust, A., Green, S., Argos, P., Kumar. V., Walter, P., Bornert, J. M., and tween regions located both in the DNA and hormone-binding ChambÃ³n, P. The chicken oestrogen receptor sequence: homology with v- domains.7 It may be speculated, therefore, that region CI of erb\ and the human oestrogen and glucocorticoid receptors. EMBO J., 5: 891-897, 1986. each ER monomer recognizes one-half of the palindromic ERE. 4. Klug, A., and Rhodes, D. "Zinc finger": a novel protein motif for nucleic Furthermore, it has been proposed that the structure of a zinc- acid recognition. TIBS 12:464-469, 1987. stabilized DNA-binding finger contains a helical region which 5. Kumar, V., Green, S., Staub, A., and ChambÃ³n, P. Localisation of the may contact DNA (14-16). The DNA-binding domain of the oestradiol binding and putative DNA binding domains of the human oestro gen receptor. EMBO J. 5: 2231-2236, 1986. nuclear receptor family may therefore be analogous to the helix- 6. Webster, N. J. G., Green, S., Jin, J. R., and ChambÃ³n, P. A hormone- turn-helix motif with region CI lying across the major groove inducible transcription activation function is present in a region of the oestrogen and glucocorticoid receptor which contains the hormone binding of the ERE making specific contacts in addition to forming domain. Cell, 54: 199-207, 1988. protein-protein contacts between two ER monomers. One in 7. Druege, P. M., Klein-Hitpass, L., Green, S., Stack, G., ChambÃ³n, P., and Ryffel, G. U. Introduction of oestrogen responsiveness into mammalian cell teresting possibility is that interaction between the two CII lines. NucÃ.Acids Res. 14:9329-9337, 1986. regions may be stabilized through the coordination of common 8. Klein-Hitpass, L., Schorpp, M., Wagner, U.. and Ryffel, G. U. An estrogen- zinc ions as has been suggested for the human immunodefi responsive element derived from the 5' flanking region of the Xenopus ciency virus Tat protein (17). If so then these zinc ions would vitellogenin A2 gene functions in transfected human cells. Cell 46: 1053- 1061, 1986. have to be coordinated tetrahedrally since extended X-ray ab 9. Cato, A. C. B., Miksicek, R., Schutz, G., Arnemann, J., and Beato, M. The sorption fine structure spectroscopy of the DNA-binding do hormone regulatory element of mouse mammary tumour virus mediates progesterone induction. EMBO J., 5: 2237-2240, 1986. main of the rat GR suggests that both zinc ions are tetrahedrally 10. Klock, G., Strahle, U., and Schutz, G. Oestrogen and glucocorticoid respon coordinated. sive elements are closely related but distinct. Nature (Lond.), 329: 734-736, 1987. It is interesting to note that of the 12 amino acids which 11. Picard, D., and Yamamoto, K. R. Two signals mediate hormone-dependent differ between the ER and GR in the 31-amino acid CI region nuclear localization of the glucocorticoid receptor. EMBO J. 6: 3333-3340, (Fig. 2) only 8 are in the zinc finger and all but 2 (Ala186,Ser201) 1987. 12. Green, S., Kumar, V., Theulaz, I., Wahli, W., and ChambÃ³n, P. The N- are nonconservative substitutions. It is therefore likely that one terminal DNA-binding "zinc finger" of the oestrogen and glucocorticoid or more of these 6 remaining amino acids (Asn189,Tyr191,Tyr"5, receptors determines target gene specificity. EMBO J., 7: 3037-3044, 1988. Trp200, Glu203,Gly204)are involved in recognizing the two base 13. Green, S., and ChambÃ³n, P. Oestradiol induction of a glucocorticoid-respon- pairs (A.T, C.G) of the ERE (TGACCT) half-palindrome with sive gene by a chimaeric receptor. Nature (Lond.), 325: 75-78, 1987. 14. Brown. R. S., and Argos, P. Fingers and helices. Nature (Lond.), 324: 215, differ from the GRE (TGTTCT) half-palindrome. The use of 1986. 15. Berg, J. M. Proposed structure for the zinc-binding domains from transcrip an ERE modified by guanosine methylation and phosphate tion factor IIIA and related proteins. Proc. Nati. Acad. Sci. USA, Â«5:99- ethylation together with receptor mutants containing point 102, 1988. mutations or deletions within the ER DNA-binding domain 16. Evans, R. M., and Hollenberg, S. M. Zinc fingers: gilt by association. Cell 52:1-3, 1988. 81. Theulaz, personal communication. 17. Frankel, A. D., Bredt, D. S., and Pabo, C. O. Tat protein from human ' L. P. Freedman, B. F. Luisi, Z. R. Korszun, R. Basavappa, P. B. Sigler, and immunodeficiency virus forms a metal linked dimer. Science (Wash. DC), K. R. Yamamoto, Nature, 334: 543-546, 1988. 240:70-73,1988.

2285s

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. Chimeric Receptors Used to Probe the DNA-binding Domain of the Estrogen and Glucocorticoid Receptors

Stephen Green and Pierre Chambon

Cancer Res 1989;49:2282s-2285s.

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