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

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Chimeric Receptors Used to Probe the DNA-Binding Domain of the Estrogen and Glucocorticoid Receptors1 (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 Chambón3 Laboratoire de GénétiqueMoléculairedesEucaryotes du CNRS, Unité184de Biologie Moléculaireetde GénieGénétiquedel'INSERM, Institut de Chimie Biologique, FacultédeMédecine,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. Chambón. Genomic organisation of receptor; hGR, human glucocorticoid receptor; CAS, cysteine cassette. the human oestrogen receptor gene, EMBO J., 7: 3385-3388, 1988. 2282s Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. NUCLEAR RECEPTOR DNA-BINDING DOMAIN 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-CAS42125° 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-TGC¡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'-KÕrT"185 487552E2GR-CAS595ER-GR-CAS reflect the close proximity of the responsive elements to the tk promoter. 302ÉÉ250 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- HS Y •¿NEH1 YA s-0 G•Y c1 Nil MUTANT STRUCTURE SEQUENCE ACTIVITY Q-*T!—¿' vDrn A.
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