The EMBO Journal vol. 10 no.7 pp. 1 875 - 1 883, 1 991

Characterization of the hormone responsive element involved in the regulation of the progesterone receptor gene

J.F.Savouret, A.Bailly, M.Misrahi, C.Rauch, G.Redeuilhl, A.Chauchereau and E.Milgrom Hornones et Reproduction and 'Communications Hormonales, Inserm Unite 33, H6pital de Bicetre, 78 rue du General Leclerc, 94275 Le Kremlin-Bicetre, France Communicated by E.Milgrom

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to - 2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, sitedirected mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+ 723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for downregulation whereas deletions of various parts of the Nterminal domain were without effect. Key words: hormone responsive elements/progesterone receptor gene/transcription regulation

Introduction The physiological action of progesterone depends on the presence of its cognate receptor in target organs. The progesterone receptor (PR) belongs to a superfamily of ligand-induced transactivators (reviewed in Green et al., 1986; Evans, 1988; Green and Chambon, 1988; Beato, 1989). They exert their regulatory activity on discrete genes through DNA binding, generally at multiple locations, on specific sequences possessing a high level of dyad symmetry. Such binding sites are termed 'Hormone Responsive Elements' or HREs (reviewed in Yamamoto, 1985; Beato, 1989). The main functions of progesterone are related to control of the menstrual cycle and pregnancy, both phasic Oxford University Press

events subject to a strict timing controlled by a multiple hormonal interplay. This situation has been shown to imply regulatory controls through modifications of receptor concentration (Milgrom et al., 1973; Bayard et al., 1978). The induction of the progesterone receptor by estrogens and its down-regulation by progestins have been demonstrated at the levels of protein and mRNA in most tissues of the genital tract of mammals and birds (Milgrom et al., 1973; Vu Hai et al., 1977; Mester and Baulieu, 1977; Horwitz and McGuire, 1978; Isomaa et al., 1979; Read et al., 1988;

Alexander et al., 1989; May et al., 1989). Cyclic variations of the progesterone receptor during the menstrual cycle and pregnancy have been described (Vu Hai et al., 1977; Bayard et al., 1978; Garcia et al., 1988). The understanding of the molecular mechanisms of regulation of PR gene transcription is thus of interest, on the one hand because they would yield information on the mechanism regulating the activity of a gene transactivator, and on the other hand because they would shed light on important regulations in reproductive biology. Moreover, inhibitory activities of nuclear receptors still remain poorly understood and the mechanism of downregulation of the progesterone receptor by its own ligand may be an interesting model in this respect, specially since ligand-induced down-regulation of receptors is a frequent feature (Milgrom et al., 1973; Dong et al., 1988; Tan et al., 1988; Berkenstam et al., 1989). It is also known that the effect of anti-estrogens is somewhat atypical in the case of the PR gene: tamoxifen being known to induce progesterone receptor synthesis in the same breast cancer cells where it completely abolishes estrogen-provoked proliferation and induction of various genes (52K, pS2/BCEI) (Horwitz and McGuire, 1978; Jordan and Prestwich, 1978; Chalbos et al., 1982; Prud'homme et al., 1985). We have used transient expression and DNase I protection by estrogen and progesterone receptors to define the HREs involved in the physiological regulation. Among several receptor binding sites present in the 5' flanking region and the beginning of the rabbit progesterone receptor gene, these methods indicated that hormonal regulation was actually due to a single ERE present on the site of initiation of translation. The properties of this ERE were compared with those of the well characterized ERE present in the Xenopus laevis vitellogenin A2 gene and shown to be completely different in respect of antiestrogen activity and progestin down-regulation.

Results A fragment of the progesterone receptor gene imparts hormonal regulation upon a heterologous structural gene

The regulatory elements of many genes are located in their 5' flanking regions. We thus initially inserted the -2762/ -90 fragment of the PR gene upstream of the 1875

J.F.Savouret et al.

thymidine kinase (TK) promoter and chloramphenicol acetyl transferase (CAT) coding sequence (+ 1 being the upmost site of initiation of transcription) (Misrahi et al., 1988). This construct was transfected in 47-D breast cancer cells either alone or in cotransfection with an expression vector encoding the human estrogen receptor (ER) (Green et al., 1986; Greene et al., 1986). Neither estrogen-induced increase nor progestin-induced decrease of transcription could be observed (Figure .1). Similar results were obtained in Cos-7 cells (data not shown). A likely explanation for this result was that the regulatory regions were located outside the tested region. Larger fragments encompassing the first exon of the PR gene were inserted in a promoterless CAT vector. We observed that hormonal regulation could be imparted to the CAT gene by a fragment (-2762/+788) of the PR gene (Figure 1). Upon cotransfection of that construct in T 47-D cells with the ER expression vector, 10 nM estradiol induced a 3.8-fold increase in CAT activity. Simultaneous administration of 10 nM of the synthetic progestin R5020 suppressed 76% of that estrogen induction and also somewhat decreased the basal level when administered alone (Figure 1). The progestin mediated down-regulation could be reversed by a 100-fold excess of the antiprogestin R38486 (1 tM), which had no effect on estrogen induction in the absence of progestins (Figure 1). The vitellogenin A2-TK-CAT (vitA2-TK-CAT) construct showed no influence of progestins on its estrogen induction in our conditions, suggesting a difference in regulatory mechanisms (Figure 1). Two incidental observations were made during this study: estrogen inducibility of the PR -CAT constructs could be observed in T 47-D cells only in cotransfection with the ER expression vector. This lack of ability of the low levels of

endogenous ER present in those cells (Horwitz et al., 1978) to regulate the transfected promoter contrasts with similar experiments using vitA2-TK-CAT which is readily inducible by endogenous ER (Savouret et al., 1990). We also observed that estrogen induction and progestin downregulation are serum dependent in T 47-D cells, but not in Cos-7 cells. When stripped fetal calf serum was used instead of stripped calf serum in T 47-D cells transfections, both phenomena were drastically decreased (data not shown). The PR gene and 5' flanking sequences contain multiple binding sites for estrogen and progesterone receptors The ability of the (-2762/+788) fragment of the PR gene to impart physiological regulation upon an heterologous gene led us to search this region for the corresponding receptor binding sites. The DNase I protection method performed on both strands detected three estrogen receptor binding sites at positions -2480/-2459, -433/-419 and +698/+723 (Figure 2). A weaker signal was also present at +750/+774 (data not shown). Two progesterone receptor binding sites were seen at positions - 1764/- 1744 and - 1077/- 1055 (Figure 2). Table I shows a comparison of the core sequence of the three estrogen receptor binding sites with the consensus sequence for EREs (Walker et al., 1984), which reveals numerous mismatches. Of particular interest is the 'T for C' mismatch in the last nucleotide of the consensus at position +718 in the third estrogen receptor binding site (see Table I): the corresponding G (in the opposite strand) has been shown to be protected by estrogen receptor contact during in vitro methylation interference experiments using the vitellogenin A2 ERE (Klein-HitpaB et al., 1986).

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Fig. 1. A fragment of the PR gene imparts hormonal regulation upon the CAT structural gene. T 47-D cells were transfected with (-2762/+788)-CAT in the presence or absence of the ER expression vector. Other constructs cotransfected with the ER expression vector were: a promoterless CAT vector, the empty TK-CAT vector, (-2762/-90)-TK-CAT and vitA2-TK-CAT. Hormonal treatments consisted of 10 nM estradiol (E), 10 nM R5020 (P) and 1 /iM RU38486 (RU486), alone or in combinations. Reversal of progestin down-regulation by RU38486 is shown for (-2762/+788)-CAT. Estrogen induction is calculated as the ratio of acetylated chloramphenicol formed in 90 min at 37°C by 100 ig of proteins from estrogen-treated cells over untreated control cells. Down-regulation is expressed as the percentage of estrogen induction that is inhibited by simultaneous progestin treatment. The numbering in the construct maps originates from the transcription start site indicated by a crooked arrow.

1876

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Determination of the estrogen receptor binding site Involved in the physiological regulation of the progesterone receptor gene 5' and 3' deletions of the progesterone receptor gene promoter and adjacent 5' non-coding region of the gene were constructed to determine which receptor binding site was responsible for the hormonal regulation. These DNA fragments were inserted in front of the promoterless CAT vector and tested by transient expression experiments (Figure 3). Deletion of nucleotides -2762 to -325 or -2762 to -165 did not decrease estrogen inducibility, showing that ER binding sites at -2480/-2459 and -433/-419 do not participate in hormonal regulation, at

least in these experimental conditions. Fragment -30/+788 retained some estrogen inducibility although this deletion markedly impairs promoter activity. Deletions were also performed starting from the 3' end of the -2762/ +788 fragment. Deletion of the +389/ +788 region decreased the basal level and no estrogen inducibility could be observed. Site-directed mutagenesis was performed to delineate further the role of the intragenic ERE by deletion of nucleotides +708 to +711. The (-2762/+788)(A + 708/ +711) - CAT construct also showed a decreased ER

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basal level of expression that was not inducible by estradiol (Figure 3). These experiments strongly suggested that the ER binding site present at position +698/+723 could mediate estrogen inducibility of the PR gene. A surprising finding was that progestin mediated down-regulation could be observed after deletion of the two PR binding sites at - 1764/- 1744 and - 1077/-1055. It was thus observed on gene fragments containing no PR binding site but only the + 698/ +723 estrogen receptor binding site. Identical results were obtained when the (- 325/+788)-CAT construct was cotransfected in Cos-7 cells together with ER and PR expression vectors (Figure 4). The hormonal effects were shown to be receptor dependent in Cos-7 cells since they were not observed when the corresponding receptor expression vector was omitted in the transfection (data not shown). A primer extension was performed to assess the accuracy of transcription initiation. Poly(A)+ RNAs were prepared from Cos-7 cells cotransfected with a (-325/+788) -Globin construct and expression vectors for ER and PR. Estrogen induced mRNAs were correctly initiated at positions (+ 1) and (+ 15) as previously described for the PR mRNA from rabbit endometrium (Misrahi et al., 1988). All specific

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Fig. 2. Estrogen and progesterone receptor binding sites in the 5' flanking and intragenic regions of the progesterone receptor gene. El: Interaction of ER with the NdeI(-2547)-PpuMI(-2008) fragment, 5' end-labelled at the NdeI site. P1: Interaction of PR with the PpuMI(-2008)-Bglll (-1601) fragment 5' end-labelled at the BgII site. P2: Interaction of PR with the BglIH(-1601)-PpuMI(-990) fragment 5' end-labelled at the PpuMI site. E2: Interaction of ER with the PpuMI(-990) -NdeI(-171) fragment 5' end-labelled at the NdeI site. E3: Interaction of ER with the HphI(+85)-BamHI(+785) fragment labelled at the BamHI site at the 3' (upper strand: U) or the 5' (lower strand: L) end. The DNase I protection experiments were performed with the indicated concentrations (in nM) of receptors. Protected sequences (bars) are positioned with respect to the initiation of transcription. G+A and T+C are base-specific sequencing reactions. 1877

9'i,F

J.F.Savouret et al.

initiations induced by estradiol were reduced by simultaneous progestin treatment (data not shown). These results strongly suggested that the ERE at position + 698/ +723 might be solely responsible for estrogen inducibility and more strikingly for progestin-mediated down-regulation although it contains no detectable PR binding site (not even GRE/PRE consensus). Studies of the estrogen responsive element of the progesterone receptor gene We therefore synthesized an oligonucleotide corresponding to the estrogen response element present at position +698/+723 and inserted it upstream of the TK promoter driving the CAT structural gene. This construct was cotransTable I. DNA sequences of the steroid receptor binding sites in the progesterone receptor gene

Estrogen receptor binding sites El (-2480/-2459) stronga E2 (-433/-419) medium a E3 (+698/+723) stronga ERE consensus sequencec Progesterone receptor binding sites P1 (-1764/-1744) mediuma P2 (-1077/-1055) stronga GRE/PRE consensus sequencee

aThe intensity

HRE core sequence

GTACA AGA TGACC GGTCA TGT CGATT GGTCG ACA TGACT b 5'-GGTCA NNN TGACC-3'

TTGCAT TTA TGTTCCd GGAACA ATC AGTTCC 5'-GGTACA NNN TGTTCT-3'

of binding estimated from the extent of protection is indicated for each site. A weak ER binding site bearing scarce homology with the consensus is located at +750/+774 and is not shown here. bT for C mismatch at position +718 is underlined. CFrom Walker et al. (1984). dThe half consensus of P1 lies on the opposite strand. eFrom Beato (1989).

fected in T 47-D cells with the ER expression vector. Estrogen inducibility was observed. The dose -response for various estradiol concentrations was very similar to that of a TK -CAT construct containing the +23/+788 fragment of the PR gene (Figure 5). This synthetic ERE was also tested for another characteristic of PR regulation: its differential response to two types of anti-estrogens, tamoxifen and ICI 164384. As shown in Table II, the synthetic ERE was regulated similarly to the +23/+788 fragment of the gene and exhibited partial induction by ER in the presence of tamoxifen, whereas ICI 164384 behaved as a complete antagonist. Strikingly, in the same conditions, a synthetic ERE from the Xenopus laevis vitellogenin A2 gene (vitA2 -TK -CAT) displayed a totally different regulation, both tamoxifen and ICI 164384 being pure antagonists (Table II). Finally, we examined the possibility that this ERE might also be responsible for another feature of progesterone receptor gene regulation: its down-regulation under the effect of progesterone. The (+23/+788)-TK-CAT construct was thus cotransfected with ER and PR expression vectors and challenged with estradiol and various concentrations of the progestin R5020 in Cos-7 cells. This treatment provoked a dose-dependent decrease of the estradiol-induced CAT activity (Figure 6), Such a regulation was also observed in T 47-D cells with (+23/+788)-TK-CAT and (+698/+729)-TK-CAT (bearing the oligonucleotide corresponding to the ERE). However, in this particular case, great care had to be taken to maintain a balance between ER and PR during transfection. Indeed, in contrast with the native promoter (Figures 1 and 3), the observance of downregulation of the TK -CAT constructs bearing the ERE upstream of the promoter (i.e. in reverse position) required the cotransfection of both expression vectors encoding ER and PR receptors. 50% down-regulation was obtained for 50 pM R5020. A 100-fold excess of RU38486 reversed the effects of R5020 on (+698/+729) -TK-CAT: down-

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Fig. 3. 5' and 3' deletion localize the regulatory region of the PR gene within the coding sequence. Various deletion constructs of the PR gene promoter were cotransfected in T 47-D cells with the ER expression vector. Cells received either estradiol 10 nM (E) or R5020 10 nm (P) or both as indicated above the CAT assays. Estrogen induction and progestin down-regulation are calculated as in Figure 1.

1878

Progesterone regulation

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87% in the presence of R5020 (10 nM) and

25% with R5020 plus 1 ItM RU38486 (Figure 7). In the same conditions, the vitA2 -TK -CAT construct exhibited :+

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(Figures 6 and 7). When (+698/+729)-TK-CAT was cotransfected into Cos-7 cells, a strong basal activity was observed in the absence of any hormonal stimulus, impeding studies of regulation (data not shown). We also synthesized oligonucleotides corresponding to the two other estrogen receptor binding sites (-2480/+2459 and -433/-419). Each one of them inserted upstream of the TK promoter was devoid of biological activity when cotransfected with the ER expression vector. Estrogen inducibility was, however, observed when they were inserted in tandem but was not repressed by progestins (data not shown). Finally, the strongest PR binding site (- 1077/- 1055) was tested in the same system and exerted a stimulatory activity in the presence of progestins. A similar positive effect was observed when using the 1871/ -810 fragment of the PR gene which contains the two PR binding sites located at -1764/- 1744 and -1077/-1055 (data not shown). -

Fig. 4. The PR gene promoter is normally regulated in Cos-7 cells. Cos-7 cells were transfected with (-325/+788)-CAT or the empty CAT vector together with ER and PR expression vectors. Transfected cells were either kept untreated (C) or received 10 nM estradiol (E) or 10 nM estradiol plus 10 nM R5020 (EP).

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The progesterone receptor does not act through a change in the affinity of ER for the ERE Down regulation of progesterone receptor gene transcription could possibly occur through two mechanisms. Either progestin-receptor complexes could decrease the affinity of ER for its ERE, or they could through a non-specified mechanism, decrease its transactivating efficiency. The first mechanism was amenable to experimental analysis: a labeled fragment of the PR gene encompassing the + 698/ +723 ERE was incubated with a non-saturating concentration of estradiol ER complexes. R5020 - PR complexes were added at various concentrations. As shown in Figure 8, no difference was observed in the binding of ER even in the presence of a 10-fold molar excess of PR. -

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Fig. 5. Estrogen inducibility of the isolated ERE at +698/+723. T 47-D cells were cotransfected with either (+23/+788)-TK-CAT (O -0) or (+698/+729)-TK-CAT ( - 0) and the ER expression vector. Transfected cells were submitted to increasing concentrations of estradiol (10-12 to 10-7 M) for 40 h. Transfections were performed in duplicate and average values are shown. Maximal estrogen stimulations are shown as CAT assays. Quantitative results are given as percentages of these maximal stimulations.

Table H. Differential effect of anti-estrogens Constructs

(+698/+729)-TK-CAT (+23/+788)-TK-CAT vitA2-TK-CAT

on

CAT E2

3.1 4.2 4.9

Progesterone receptor domains involved in the down-regulation of PR gene transcription To determine the receptor regions involved in the downregulation mechanism, we replaced the wild type progesterone receptor by deletion mutants in transfection experiments. Western blot analysis with anti-PR monoclonal antibodies (Logeat et al., 1985) showed that all expression vectors produced equivalent amounts of truncated progesterone receptor in transfected cells (data not shown). As shown in Table III, progesterone receptors deletion in the steroid binding region were inactive. Mutants A662-930 was specially interesting since it has been shown to exert a constitutive stimulatory activity on some positively regulated genes such as the promoter in the Long Terminal Repeat of the Mouse Mammary Tumor Virus (MMTV-LTR) (Guiochon-Mantel et al., 1988). Deletions in the N-terminal part of the proteins A 104-371 and A372-543 were without distinctive effect on down-regulation ability whereas

estrogen induction of PR gene constructs in T 47-D cells

activity (induction factor)a tamoxifen

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1.6

3.4 2.5 0.7

0.4 0.8 0.1

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aCalculated as in Figure 1. Data are the mean of duplicate experiments performed on two cell passages. T 47-D cells were cotransfected with the different constructs plus the ER expression vector, then received 0.1 nM E2, 100 nM tamoxifen, 100 nM ICI 164384, alone or combined as shown. 1879

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J.F.Savouret et al.

deletions of the DNA binding zinc fingers in A546-593 and A592 -641 abolished the activity. Again a difference was observed with the stimulatory activity on the MMTV-LTR since the deletion mutant A104-371 is only 5% active on the latter whereas it is fully active in down-regulation.

Discussion Topology of hormone responsive elements in the

rabbit progesterone receptor gene promoter Previous studies had shown the presence in the 5' flanking region of the rabbit progesterone receptor gene (Misrahi et al., 1988) and inside the gene (J.F.Savouret, unpublished data) of several sequences homologous to the consensus for estrogen and progesterone responsive elements. DNase I protection experiments performed in the present study showed that only three of them did indeed bind estrogen receptor and two did bind the progesterone receptor.

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Fig. 6. Progestin inhibition of estradiol-mediated induction of the PR

gene transcription in Cos-7 cells. Cos-7 cells were cotransfected with the (+23/+788)-TK-CAT construct and ER and PR expression vectors. Cells were submitted to 10 nM estradiol and challenged with increasing concentrations of R5020 (10-12 to 10-7 M). Average values of duplicate experiments are shown (0 - 0). The curve shows the decrease of estradiol induction provoked by R5020, expressed as percentage of down-regulation (see Figure 1). Also shown are CAT assays from transfections performed with (+23/+788)-TK-CAT, vitA2-TK-CAT and the TK-CAT vector. Cells received 10 nM estradiol (E), 10 nM R5020 (P) or a combination of both. Downregulation was 77.5% for (+23/+788)-TK-CAT and 0% for the vitA2 -TK-CAT construct.

Fig. 8. The progesterone receptor shows no influence on the affinity of ER for its ERE at +698/+723. An EcoRI-BstXI fragment containing sequences from (+588) to (+877) of the PR gene was 5' end-labelled at the BstXI site and incubated without (-) or with (+) 5.5 nM of ER in the presence of various concentrations of PR (nM) as indicated. The position of the ER binding site overlapping the ATG for initiation of translation is shown.

Table HI.

Progesterone receptor domains involved in PR gene downregulation at the transcriptional level Wild-type or mutated Progestin down-regulationa progesterone receptor

(%)

wild-type

75.6 + 5.5 76.3 59.5