MOLECULAR AND CELLULAR BIOLOGY, Dec. 1988, p. 5323-53300270-7306/88/125323-08$02.00/0Copyright C) 1988, American Society for Microbiology

Estrogen and Progesterone Receptor-Binding Sites on the ChickenVitellogenin II Gene: Synergism of Steroid Hormone ActionANDREW C. B. CATO,'* ELLEN HEITLINGER,1t HELMUT PONTA,' LUDGER KLEIN-HITPASS,lt

GERHART U. RYFFEL,1 ALAIN BAILLY,2 CLAUDINE RAUCH,2 AND EDWIN MILGROM2

Kernforschungszentrum Karlsruhe, Institut fur Genetik und Toxikologie, Postfach 3640, D-7500 Karlsruhe 1, FederalRepublic of Germany,1 and Unite de Recherches Hormones et Reproduction, Institut National de la Sante et de la

Recherche Medicale, Unite 135, Faculte de Medecine Paris-Sud, 94275 Le Kremlin-Bicetre Cedex, France2

Received 21 June 1988/Accepted 19 September 1988

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfectionexperiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin,and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNAsequences from -626 to -590 upstream of the start of transcription of the chicken viteliogenin gene linked tothe herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicolacetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and theprogestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergisticaction of these two steroids. This synergism was not observed upon treatment of the transfected cells withestradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we

showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, forthe synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to thevitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to theconsensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate herethat two different steroid hormones can be functionally synergistic through the interaction of their correspond-ing receptors with two different binding sites adjacent to one another.

The regulation of gene expression by steroid hormones isthought to occur through the interaction of steroid hormonereceptor complexes with discrete nucleotide sequences inthe neighborhood of the promoters of inducible genes (forreviews, see references 2 and 35). Short perfect or imperfectpalindromic sequences that mediate steroid hormone actionhave been identified near the promoters of a number ofsteroid hormone-regulated genes. For example, 13-base-pair(bp) palindromic elements that mediate the estrogen re-

sponse have been identified near the promoters of theXenopus and chicken vitellogenin genes (4, 17, 18, 19, 22).Similarly, 15-bp perfect or imperfect palindromes that en-

compass the hexanucleotide motif 5'-TGTT/CCT-3' havebeen shown to mediate glucocorticoid and progestin re-

sponses in a number of genes (5, 6, 29, 30). The delimitationof these short nucleotide sequences was achieved throughgene transfer studies with either in vitro mutagenesis ofknown hormone receptor-binding sites (6) or different chi-meras containing synthetic oligonucleotides (13, 17, 30). In a

number of hormone response elements, such as that inmouse mammary tumor virus DNA (3, 6), the tyrosineaminotransferase gene (13), and the Xenopus vitellogenin Biand chicken vitellogenin II (VTG II) genes (4, 16, 22, 27),multiple copies of the same sequence motif are present. Insuch cases, steroid hormone response is achieved by a

concerted action of the individual motifs. In some othergenes, such as the tryptophan oxygenase gene (8) or mouse

mammary tumor virus DNA (6, 24), the receptor-binding

* Corresponding author.t Present address: Biozentrum, CH-4056 Basel, Switzerland.t Present address: Department of Cell Biology, Baylor College of

Medicine, Texas Medical Center, Houston, TX 77030.

sites function by cooperating with the binding sites for othertranscriptional factors that lie in their immediate vicinity.

In the VTG II gene, a gene whose transcription is knownto be regulated by estradiol, two regions conferring theestrogen response have been identified (4, 17). The distalregion that stretches from -626 to -613 5' upstream of thestart of transcription of this gene contains a single perfectcopy of the dyad element 5'-GGTCANNNTGACC-3',whereas the proximal region contains two imperfect copiesof this element (4). The distal estrogen response element(ERED) has been shown to bind the estrogen receptor withhigh affinity (17, 25), and it is assumed from receptor-bindingstudies with mutated estrogen response elements (EREs)(17, 23) that the proximal elements may also bind theestrogen receptor but with greatly reduced affinity. Close tothe center of symmetry of the ERED is the conservedhexanucleotide 5'-TGTTCT-3' on the lower (or antisensestrand), which is the core motif for binding to DNA by theglucocorticoid receptor. In vitro receptor-DNA bindingstudies have shown that this hexanucleotide and neighboringsequences on the VTG II gene are bound by the glucocorti-coid receptor (26) and could also possibly be recognized bythe androgen receptor (6). The hexanucleotide motif is alsopart of the recognition sequence of the progesterone recep-tor (1, 14, 29).The close proximity of the hexanucleotide motif to the

ERED on the VTG II gene prompted us to study thefunctional interaction of these two elements with progestin,androgens, and estradiol in transfection experiments inT47D cells that contain receptors for all three steroid hor-mones (6). In this communication we present the results ofthese studies.

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5324 CATO ET AL.

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FIG. 1. Chimeric chicken VTG Il-TK-CAT constructs. Oligonucleotide sequences corresponding to the region between -626 and -590of the chicken VTG II gene were synthesized and cloned in front of the herpes simplex virus TK promoter (-105/+51) driving thetranscription of the bacterial CAT gene (pERE+PRE+). The sequence of the VTG II gene fragment is indicated. In the other constructs,pERE+PRE-, pERE-PRE+, and pERE-PRE-, point mutations were introduced into the VTG II gene fragment. The dotted lines representDNA sequences exactly as they occur in the vitellogenin gene fragment, and the base alterations are indicated. The horizontal arrows indicatethe palindromic sequence that mediates the estrogen response and binds the estrogen receptor (17, 25). The boxed sequence is the conservedhexanucleotide motif 5'-TGTTCT-3' (in the reversed orientation) shown to bind the glucocorticoid receptor (26). tk = TK.

MATERIALS AND METHODS RESULTS

Plasmid construction. DNA sequences derived from theVTG II gene were synthesized with the Pharmacia GeneAssembler machine. The fragments were synthesized withHindIlI and BamHI linkers at their 5' and 3' ends, re-spectively. The oligonucleotides were then cloned into theHindIII-BamHI site of the plasmid vector pBL-CAT8+ aspreviously described (17).

Cell culture and transient transfection. Human T47D cellswere cultured in RPMI 1640 medium supplemented with 10%fetal calf serum and 0.6 ,ug of insulin per ml. Two days priorto transfection, the cells were washed once with phosphate-buffered saline and cultured in Dulbecco medium withoutphenol red (GIBCO Laboratories and Bethesda ResearchLaboratories, Inc.) and supplemented with 10% fetal calfserum (stripped of hormone by charcoal treatment [34]) and0.6 ,ug of insulin per ml. The cells were cultured in thismedium during the transfection procedure and subsequentsteps. Transfection was carried out by the DEAE-dextranprocedure as previously described (5). Concentrations ofsteroids administered to the cells were S x 10-8 M 17-p-estradiol (E2), 10-8 M progestin R5020, and 10- M dihy-drotestosterone (DHT). The transfected cells were treatedwith or without hormone, harvested, and treated further forassay of chloramphenicol acetyltransferase (CAT) activityas previously described (5).

Progesterone receptor-DNA interaction. The progesteronereceptor, complexed to [3H]16 a-ethyl-21-hydroxy-19 nor-pregna-4-en-3, 20-dione (Amersham Corp.), was purifiedfrom rabbit uterus by immunoaffinity chromatography asdescribed previously (20). DNase I footprinting with thispurified receptor and 32P-end-labeled fragments of con-structs of pERE+PRE+, pERE-PRE+, and pERE+PRE-was carried out as previously described (1), except that theincubation was done in a 34-,ul volume before the addition ofDNase I (2 [lI of a 3.6-,ug/ml solution).

Transcription analyses by primer extension. Polyadenyl-ated RNAs were extracted from T47D cells cotransfectedwith the construct pERE+PRE+ and ptk4*CAT3 (5). TheRNAs were hybridized to a 5'-end-labeled primer corre-sponding to the first 30 coding nucleotides of the CAT gene(3'-TACCTCTTTTTTTAGTGACCTATATGGTGG-5').The primer extension reaction was carried out essentially asdescribed previously (22), with the exception that avianmyeloblastosis virus reverse transcriptase (Life Sciences)rather than Moloney murine leukemia virus reverse tran-scriptase was used. The primer extension reaction wascarried out at 42°C.

To study the functional significance of the estrogen andputative progesterone receptor-binding sites on the VTG IIgene, we used a 37-bp oligonucleotide corresponding to thesequence from -626 to -590 of the VTG II gene linked tothe thymidine kinase (TK) promoter (-105 to +51) coupledto the bacterial CAT gene (pERE+PRE+, Fig. 1). This VTGII chimeric construct was transiently transfected into thehuman mammary tumor cell line, T47D, that contains andro-gen, estrogen, and progesterone receptors, and the transfec-ted cells were treated with either E2 or the progestin R5020.Upon assay of CAT activity, we observed that E2 inducedCAT activity 12-fold in the transfected cells (pERE+PRE+,Fig. 2A) and that R5020 induced CAT activity 8-fold(pERE+PRE+, Fig. 2A). In contrast, the TK promoter andCAT sequences alone without the VTG II gene fragmentshowed no response to either E2 or R5020 (pBLCAT8+, Fig.2A). Thus, the cloned 37-bp VTG II gene fragment mustcontain a progestin response element (PRE) in addition tothe previously described ERE (4, 17).The ERED and surrounding sequences on the VTG II gene

fragment had been shown previously to be involved inestrogen receptor binding (15, 25). To determine whichsequence on this fragment binds the progesterone receptor,we used a progesterone receptor purified from rabbit uterusin DNase I footprinting experiments with the VTG II genefragment from the construct pERE+PRE+ (Fig. 3). Se-quences containing the conserved hexanucleotide motifwere protected against DNase I digestion by the receptor(Fig. 3). The limits of the protected region on the VTG IIgene fragment were -610 and -590. There were specificbases in the footprint region that were strongly or lessstrongly protected against DNase I cleavage. For example,the A residue at position -599 on the upper DNA strand(Fig. 3B and C) or the G and C residues at positions -600and -603 on the lower strand (Fig. 3A and C) were stronglyprotected against DNase I cleavage by the receptor. Thisresult indicates that these base residues are important con-tact points for interaction of the progesterone receptor in itsbinding to the VTG II DNA. The palindromic sequenceERED required for estrogen receptor binding and estradiolregulation was not protected against DNase I digestion bythe progesterone receptor (Fig. 3).

Binding of the progesterone receptor to its cognate bindingsite generates alterations in the DNA structure at its bound-aries, as evidenced by an enhanced cleavage of these sites byDNase I. Such DNase I-hypersensitive sites have beenobserved at the boundaries of the footprint region of the

MOL. CELL. BIOL.

SYNERGISM OF ESTROGEN AND PROGESTERONE RESPONSES

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pBL CAT 8+ pERE+ PRE pERE+ PRE- pERE- PRE pERE- PRE- pBL CAT8+ pERE+PRE+FIG. 2. Progestin, androgen, and estradiol induction of CAT activity in T47D cells transiently transfected with the VTG II-TK-CAT

constructs. The VTG II-TK-CAT constructs and the vector pBLCAT8+ (17) were separately transfected into T47D cells that had beenpreviously cultured in Dulbecco medium without phenol red and supplemented with 10%o fetal calf serum (stripped of hormone by charcoaltreatment [34]) (see Materials and Methods). (A) The transfected cells were treated without hormones (-), with 5 X 10-8 M E2 (E2), with 10-8M R5020 (R), or with a combination of E2 and R5020 (E2 + R). (B) The transfected cells were treated without hormones (-), with 5 x 10-8M E2 (E2), with 10-7 M DHT (D), or with a combination of E2 and DHT (E2 + D). CAT activities measured in the transfected cells arepresented as bars. Each bar represents the average result of four different experiments carried out with at least two independent plasmid DNApreparations. The degrees of variation of the individual results are indicated.

progesterone receptor on other genes (1, 14, 29). On theVTG II gene fragment, the DNase I-hypersensitive siteswere localized at positions -620 and -612 to -615 on thelower DNA strand (Fig. 3A and C). Other sites were at the 5'end of the TK promoter of the construct pERE+PRE+ (Fig.3B and C). As the nucleotide sequences at positions -620and -615 are within the palindromic sequence of the ERED,we assumed that the binding of the progesterone receptor tothe VTG II gene fragment would alter the functional activityof the ERE.To investigate this idea, we added E2 and R5020 simulta-

neously to T47D cells transfected with the pERE+PRE+construct. This combination of steroids resulted in a 50-foldinduction of CAT activity (pERE+PRE+, Fig. 2A), a valuemuch higher than the additive effects of E2 and R5020 (Fig.2A). This superinduction of CAT activity which we refer toas synergistic action of the two steroids indicates that thesimultaneous interaction of the progesterone and estrogenreceptors with the VTG II gene fragment causes a substan-tial change in hormone response mediated by this piece ofDNA.To determine the doses of the two steroids required for

this synergism, we transiently transfected the construct(pERE+PRE+) into T47D cells and treated the transfectedcells with different concentrations of either E2 (Fig. 4A) or

R5020 (Fig. 4B). E2 induced CAT activity dose dependentlyin the T47D cells with a half-maximal concentration of 0.02nM (Fig. 4A). R5020, on the other hand, induced CATactivity with a half-maximal concentration of 0.2 nM (Fig.4B). When 0.02 nM E2 was added to the transfected cells andthe concentrations of added R5020 were altered (+E2, Fig.4B), higher levels ofCAT activity were observed. This resultagrees with our finding (Fig. 2A) that E2 acts synergisticallywith R5020 in the induction ofCAT activity in pERE+PRE+-transfected T47D cells. However, the concentration of

R5020 required for half-maximal induction in the presence ofE2 was identical to that required in the absence of E2 (Fig.4B). This result indicates that the interaction of the proges-terone receptor with the VTG II fragment is unaffected bythe binding of the estrogen receptor to the same fragment.On the contrary, when the transfected cells were treatedwith the half-maximal concentration of R5020 (0.2 nM) andvarious concentrations of E2, the dose-dependent curve ofE2 was shifted to the left (+R5020, Fig. 4A). This shift in thedose-dependent curve of E2 might indicate an altered affinityof the estrogen receptor for its binding site upon binding bythe progesterone receptor. Obviously, such an alteration inthe affinity with which the progesterone receptor binds to itscognate binding site does not occur upon estrogen receptorbinding, as the dose response curve of R5020 is not altered inthe presence of E2.We have already shown that the progesterone receptor

strongly protects certain DNA residues against DNase Icleavage. We reasoned that these residues are importantcontact points for the progesterone receptor. To confirm thisfunction, we synthesized oligonucleotides corresponding topositions -626 to -590 of the VTG II gene and introduced asingle-base alteration at one of the sites contacted by theprogesterone receptors: a G residue at position -600 (on thelower strand) (Fig. 3C) was converted into a T residue. Theresulting oligonucleotide was cloned into the pBLCAT8+vector exactly as described for pERE+PRE+. This newconstruct, designated pERE+PRE- (Fig. 1), was used inDNase I footprinting experiments with the purified proges-terone receptor. The construct pERE+PRE- did not bindthe progesterone receptor (Fig. 5A). Besides, the alterationin the DNA structure observed at the ERE after progester-one receptor binding was not evident. In transfection exper-iments, construct pERE+PRE- did not mediate the pro-gestin response, although this construct clearly mediated an

VOL. 8, 1988 5325

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5326 CATO ET AL. MOL. CELL. BIOL.

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FIG. 3. DNase I footprints of progesterone receptor binding to the chicken VTG II DNA. The construct pERE'PRE' was digested withPvuIl and Bglll to generate a 319-bp fragment consisting of the VTG II DNA with neighboring vector sequences and the TK promoterelement. This fragment was labeled at the Bglll site either at the 5' end or at the 3' end for footprinting the lower or upper DNA strand,respectively (panels A and B). The labeled 319-bp fragment (2 ng) was incubated with three different progesterone receptor (PR)concentrations (25, 50, and 100 nM) or without any receptor and then digested with DNase I (see Materials and Methods). The DNAse Idigestion products were run on 6% sequencing gels. The positions of the ERED, the BamHl linker, and the TK promoter fragment (tk) areindicated. The positions of some bases that are protected against DNase I cleavage by the receptor are indicated. The arrows show theposition of bases with enhanced DNase I cleavage. The G + A and T + C lanes are sequence ladders of the same labeled DNA fragment usedfor the footprint reaction. The DNase I cleavage pattern of the VTG II fragment and neighboring sequences in the presence of theprogesterone receptor is shown schematically in panel C. The closed circles represent bases that are protected against DNase I digestion bythe receptor. The open circles represent bases with an enhanced DNase I cleavage pattern after receptor binding. Bases with double or triplecircles show a higher intensity of DNase I cleavage or protection against DNase I cleavage. The intensity maps were derived by visualinspection of the DNase I cleavage pattern. The horizontal arrows indicate the position of the 13-bp palindromic element (ERED) involvedin estrogen receptor binding and induction of transcription by estradiol (4, 17). The hatched bar indicates the area of the progesterone receptorfootprint on the VTG II DNA, and the brackets show the limits of the footprint on the upper and lower DNA strands. tk = TK.

SYNERGISM OF ESTROGEN AND PROGESTERONE RESPONSES

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estradiol and progestin induction of CAT activity in T47D cellstransfected with the pERE+PRE+ construct. The constructpERE+PRE+ was transfected transiently into T47D cells andtreated separately with different concentrations of E2 (A) or R5020(B). CAT activity, determined in nanomoles per hour per milligramof protein, was plotted graphically against the concentrations ofhormones used. The transfected cells were then treated with 0.2 nMR5020 and different concentrations of E2 (A, +R5020) or with 0.02nM E2 and different concentrations of R5020 (B, + E2). CAT activityfrom extracts of transfected cells was computed as previouslydescribed (5).

estradiol response (pERE+PRE-, Fig. 2A). The E2 responseof pERE+PRE- was, however, only 50% the E2 response ofpERE+PRE+ (Fig. 2A). In cells transfected with pERE+PRE- and treated with both E2 and R5020, the synergisticaction of the two steroids was not observed (Fig. 2A). Theseresults show firstly that position -600 on the VTG IIfragment is essential for progesterone receptor binding andfunction. Secondly, the C residue at this position is neces-

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FIG. 5. DNase I footprints of the progesterone receptor andmutant VTG II-TK-CAT constructs. The mutant VTG II-TK-CATconstructs pERE+PRE- and pERE-PRE+ were 5' end labeled atthe BglII site (+51) of the TK promoter and further cleaved withPvuII to generate a 319-bp fragment. This labeled fragment (2 ng)was incubated with 25 nM purified progesterone receptor or withoutthe receptor and then digested with DNase I. The DNase I digestionproducts were run on 6% sequencing gels. The autoradiographshows the DNase I digestion products of the VTG II fragment ofconstructs pERE+PRE- (A) and pERE-PRE+ (B) after incubationwithout (0) or with (25 nM) the progesterone receptor. The positionsof some of the bases protected against DNase I cleavage or showingenhanced DNase I cleavage after incubation with the receptor are

indicated as in Fig. 3.

sary for the efficient function of the ERE in the absence ofprogestin, as its alteration leads to a lower estrogen re-

sponse. Thirdly, the above-described results demonstratethat for the synergistic action of E2 and R5020 to occur,functional progesterone receptor binding is required.To determine the role of the ERE in the synergism, we

introduced a mutation that alters the A residue at position 11of this 13-bp palindrome into a G residue. Such a mutationdrastically reduces estrogen receptor binding and the abilityof the ERE to mediate the estradiol response (17). Intransfection experiments with T47D cells and the new con-

struct, pERE-PRE', E2 induced CAT activity only 3-fold,whereas R5020 induced CAT activity 12-fold (pERE-PRE+,Fig. 2A). In footprinting experiments, the pERE-PRE+construct was recognized by the progesterone receptor with

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VOL. 8, 1988 5327

5328 CATO ET AL.

all of the characteristic features observed in its interactionwith the wild-type construct pERE+PRE+ (Fig. 5B). TheDNase I-hypersensitive sites at the ERE observed on theconstruct pERE+PRE+ (Fig. 3A) following progesteronereceptor binding were identical to those observed on theconstruct pERE-PRE+ (Fig. SB). We would therefore ex-pect synergistic action of E2 and R5020 in the induction ofCAT activity in cells transfected with construct pERE-PRE' when treated with the two steroids. This was the case,except that the magnitude of the combined effects of E2 andR5020 was lower in cells transfected with pERE-PRE+ thanin cells transfected with pERE+PRE+ (Fig. 2A). This resultwas most likely due to the reduced binding of the estrogenreceptor to the mutated ERE on pERE-PRE+ and to thereduced estrogen response of this construct (pERE-PRE+,Fig. 2).When the point mutations in pERE+PRE- and pERE-

PRE' were introduced together, the resulting construct witha double point mutation, pERE-PRE- (Fig. 1), no longerresponded to either E2 or R5020 (pERE-PRE-, Fig. 2A).More importantly, combined treatment with the two steroidswas unable to induce CAT activity in cells transfected withpERE-PRE-. The pERE-PRE- construct, unlike thepERE-PRE+ construct, did not respond to E2 because ofthe additional mutation in the PRE at position -600 (Fig.2A), confirming our finding that the intact PRE in theabsence of progestin contributes to the estradiol response ofconstruct pERE-PRE+.We have previously shown that the correct start site of

transcription at the TK promoter of the construct pERE+PRE' is used in transfected MCF-7 cells and that E2increases that amount ofRNA initiating at this start site (17).Primer extension studies showed that the synergistic actionof E2 and R5020 that we have reported in the CAT assayreflects correct transcription at the TK promoter of con-struct pERE+PRE+ (Fig. 6).The synergism that we observed between E2 and R5020 in

the induction of CAT activity in cells transfected withpERE+PRE+ could be mimicked with E2 and the glucocor-ticoid dexamethasone instead of the progestin R5020 (resultsnot shown). This result is consistent with the finding that theglucocorticoid receptor binds the same sequences on theVTG II gene fragment that we have shown to be bound bythe progesterone receptor (26). The androgen DHT alsoinduces CAT activity in T47D cells transfected with theconstruct pERE+PRE+ (pERE+PRE+, Fig. 2B). This resultis also in agreement with the finding that the DNA-bindingdomain of the androgen receptor is 79% homologous at theamino acid level to a comparable domain of the progesteronereceptor (7, 21). However, when we treated the transfectedcells simultaneously with E2 and DHT, we did not observethe synergistic steroid hormone action which we found withE2 and R5020 (pERE+PRE+, compare Fig. 2A and B). Thus,the synergistic action of the different steroids that we havedescribed here is not a general feature of all of the steroidhormones that can induce CAT activity in our pERE+PRE+-transfected T47D cells.

DISCUSSION

We have demonstrated that the distal palindromic elementin the VTG II gene that has been identified in experiments inMCF-7 cells (17, 18) and HEPG2 cells (4) as an ERE alsomediates estrogen action in T47D cells. Estrogen receptorsin T47D cells are thought to have anomalous properties (12);however, our functional results with the ERE of the VTG II

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FIG. 6. CAT activity corresponding to correctly initiated tran-scripts at the TK promoter. A primer extension reaction was carriedout with 2 ,ug of polyadenylated RNA from T47D cells cotransfectedwith 5 ,ug of pERE+PRE+ DNA and 5 ,ug of ptkqCAT3 (5). Theprimer extension products were separated on a 6% denaturingpolyacrylamide gel. The arrow at 125nt represent transcripts origi-nating at the correct TK promoter, and the arrow at 94nt representstranscripts originating at the TK promoter of the control plasmidptkqiCAT3. This plasmid was introduced as a control for transfec-tion efficiency and does not respond to steroids. The RNAs wereextracted from transfected cells treated without hormone (-), withR5020 (+R), with E2 (+E2), or with a combination of R5020 and E2(+R+E2).

gene indicate that the estrogen receptor in these cells isnormal in that it can mediate estrogen activation of areporter gene.The VTG II gene fragment also mediates the progestin

response through the binding of the progesterone receptor toa partial palindromic sequence 5'-GGATCANNNTGTTCT-3' (the PRE). This element is homologous to the 13-bp intactpalindrome 5'-GGTCANNNTGACC-3' (the ERE) that me-diates the estradiol response. In fact, the ERE only differsfrom the PRE at three nucleotide residues. We have dem-onstrated in this work that a mutation that destroys the PREof the VTG II chimeric construct decreases the estradiolresponse of this construct (compare estradiol responses ofconstructs pERE+PRE+ and pERE+PRE- in Fig. 2A),although the PRE on its own cannot mediate an estrogenresponse (19). It is possible that the estrogen receptor bindsweakly to the PRE, which shares a great deal of sequencerelatedness with the ERE. The interaction of the estrogenreceptor that binds to the ERE and the weak interaction ofthe estrogen receptor with the PRE may be responsible forthe higher estradiol response of construct pERE+PRE+ thanof construct pERE+PRE- (Fig. 2A). Such synergistic actionof two adjacently positioned estrogen receptor-binding sitesthat are on their own inactive has been previously demon-strated (16). What is novel in the present work is that one ofthese two receptor-binding sites is not a typical ERE but aPRE. Thus, the conservation of sequences in the DNA-binding domains already described for different steroid hor-mone receptors (10) is also observed on the level of the DNAsequences that these receptors recognize.We have shown that the progesterone receptor binds to

the region from -610 to -590 upstream of the VTG II genefragment. This region includes the sequence protected by theglucocorticoid receptor (-608 to -587) (26). The contact

MOL. CELL. BIOL.

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SYNERGISM OF ESTROGEN AND PROGESTERONE RESPONSES

points of the progesterone receptor on the vitellogeninsequence involve the G and C residues of the hexanucleotide5'-TGTTCT-3' that are also required for glucocorticoidreceptor binding (26). Thus, as in other genes (14, 29, 33), theprogesterone receptor-binding site cannot be distinguishedfrom the glucocorticoid receptor-binding site.When administered together to T47D cells transfected

with the chimeric VTG II-TK-CAT construct (pERE+PRE'), estradiol and progestin acted synergistically in in-ducing CAT activity. It is not likely that the weak binding ofthe estrogen receptor at the PRE that we have suggestedcould lead to a formation of heterodimers between theestrogen and progesterone receptors at the PRE for thesynergistic steroid hormone action. The PRE alone withoutthe ERE cannot mediate a synergistic response. In experi-ments in which we transfected chimeric constructs contain-ing the PRE from the mouse mammary tumor virus hormoneresponse element into T47D cells, no synergistic hormoneaction was observed upon treatment of the transfected cellswith E2 and R5020 (J. Weinman and A. C. B. Cato, unpub-lished results). It could be argued that the synergistic actionof E2 and R5020 in cells transfected with the VTG II-TK-CAT construct is due to an overproduction of the progester-one receptor by E2 treatment, as the progesterone receptoris an estrogen-inducible protein (11) which, when induced,could lead to the higher progestin response of our con-structs. However, although estradiol can induce progester-one receptor levels in a number of tissues and cells (9, 11,28), the priming effect of estrogen cannot be used to explainour results in T47D cells. Firstly, these cells constitutivelyexpress the progesterone receptor and cannot be primed(12). Secondly, glucocorticoids, when administered togetherwith estradiol in pERE+PRE+-transfected MCF-7 cells, actsynergistically in their responses, although glucocorticoidreceptor levels are not influenced by estradiol treatment (E.Heitlinger and A. C. B. Cato, unpublished results). Ittherefore appears that the levels of the receptors are notresponsible for the synergistic action of the different ste-roids. The androgen DHT also induces CAT activity in T47Dcells transfected with pERE+PRE+ (Fig. 2B). However,estradiol does not act in synergy with DHT in the inductionof CAT activity in these transfected cells. The synergisticaction of the different steroid hormones may therefore bedue to distinct structural features of the receptors that bindthese steroids. We do not know what these features of thereceptors are, nor do we know the biological significance ofthe synergistic action of estradiol and the other steroidhormones in the expression of the chicken VTG II gene.However, from the magnitude of the response of constructpERE+PRE+ to the combination of E2 and R5020 (Fig. 2A),we believe that this synergism may play some role in theregulation of this gene.On the grounds of the sheer proximity of the ERED to the

PRE on the VTG II gene fragment, one would speculate thatprotein-protein interactions between the two receptors whenbound to their cognate binding sites may be responsible forthe synergistic action of the two steroids. A number offindings favor such speculations. Firstly, when bound toDNA, steroid hormone receptors still have the ability tointeract with each other, as revealed by electron microscopicstudies (31). Secondly, different progesterone receptor ami-no-terminal mutants have been shown to interact differentlywith the estrogen receptor in the regulation of transcriptionat the chicken ovalbumin promoter (32). Thus, the N-terminal end of the progesterone receptor could also beinvolved in the synergistic action of these two steroids on the

VTG II-TK-CAT chimeric construct. Alternatively, it ispossible that protein-protein interactions are not at all in-volved in the synergistic action of the two steroids that wehave documented in this work but that binding to the VTG IIgene fragment by one of the two receptors causes confor-mational changes in the neighboring DNA sequence in sucha way that the binding properties of the other receptor isaffected. In the study with the pERE+PRE+ construct, wehave shown that the binding of the progesterone receptor tothe VTG II gene fragment causes structural alterations in theDNA sequence of the ERE that may account for the alteredsensitivity at the TK promoter of our construct to estradiol.Whether this process of structural alteration in the EREDsequence alone is responsible for the synergistic action of E2and R5020 or whether this process also requires a protein-protein interaction between the two receptors is at presentnot known. In summary, our results show that two differentsteroid hormones can functionally cooperate in transcrip-tional activation through the binding of their correspondingreceptors to two closely adjacent receptor-binding sites.

ACKNOWLEDGMENTS

We thank D. Henderson (Berlin) for the gift of R5020 and J.Weinmann and U. Rahmsdorf (Karlsruhe) for their excellent tech-nical assistance.

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