Characterization of estrogen receptor cDNAs from human uterus: identification of a novel PvuII polymorphism

MOlecular and Cellular

ELSEVIER

EncJ=indogy

Molecular and Cellular Endocrinology 101 (1994) 101-110

Characterization of estrogen receptor cDNAs from human uterus: identification of a novel PvuII polymorphism Yaolin Wang, Richard

J. Miksicek

*

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA

(Received 8 November 1993; accepted 3 December 1993)

Abstract Using reverse transcription and the polymerase chain reaction, we have cloned estrogen receptor complementary DNAs from normal human uterine tissue. Restriction endonuclease analysis identified a polymorphic PuuII recognition site within half of the receptor cDNAs. Sequence analysis revealed a number of differences with the sequence previously reported for the ER cDNA isolated from MCF7 cells and confirmed that the codon for amino acid 400 was erroneously assigned as valine (GTG) rather than glycine (GGG). Sequencing also defined the nature of the PuuII polymorphism, with allele A coding for G1u22 and allele B (with an additional PuuII site) coding for Gln2’. We demonstrate that both alleles of this receptor activate transcription of an estrogen-responsive gene to the same extent. This selective cloning method should have wide application in the investigation of naturally occurring cDNA variants from diseased tissues, such as breast cancer cell lines and primary tumor specimens. Key words:

Estrogen

receptor;

cDNA

cloning;

DNA sequence

1. Introduction

Nuclear hormone receptors comprise a large family of proteins that mediate many important developmental events and cellular responses (Evans, 1988; CasonJurica et al., 1990; Wa.hli and Martinez, 1991). This family of receptors functions as transcription factors of which the activity depends on the binding of an appropriate hormonal ligand. In contrast to receptors for most peptide hormones and growth factors which are located on the cell surface, the nuclear receptors are present within the cells in close association with chromatin (Wrenn and Katzenellenbogen, 1990). The hormonal modulators of the nuclear receptors, illustrated most vividly by the steroid hormones, act to influence the behavior of many tissues in virtually all metazoan species, from invertebrates to mammals. Among them, estrogens play an important role in both normal reproductive physiology and in a variety of human diseases including breast cancer, osteoporosis, and cardiovascular disease. Prior to this study, the human estrogen

polymorphism;

Uterus;

Steroid

receptor

receptor (hERI cDNA was isolated and sequenced (Green et al., 1986; Greene et al., 1986) and detailed functional analysis demonstrated that this receptor consists of discrete regions responsible for DNA- and hormone-binding (Kumar et al., 19861, as well as elements for transcriptional activation (Tora et al., 1989a) and dimerization (Fawell et al., 1990). This hER cDNA, however, was cloned from a human breast cancer cell line (MCF7) that might possess an ER which differs from the ‘authentic’ receptor. Therefore we considered it important to isolate a wild-type estrogen receptor (wt-hER) cDNA from a normal human tissue in order to fully understand the role of estrogen receptor in both physiological and pathological conditions. Here we describe the cloning and characterization of non-mutated hER cDNAs from human myometrium using reverse transcription and the polymerase chain reaction. 2. Materials and methods 2.1. RNA preparation and cDNA synthesis

* Corresponding author. Tel.: (.516)-444-3054; Fax: (.516)-444-3218; Email: [email protected]. 0303-7207/94/$07.00 0 1994 El sevier Science SSDI 0303-7207(93)E0330-W

Ireland

Total RNA was isolated from frozen tissues using the method of guanidinium thiocyanate (Chomczynski

Ltd. All rights reserved

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und

and Sacchi, 1987). The specimen used for RNA preparation consisted of myometrial tissue from a hysterectomy sample performed as a result of a benign uterine fibroid. Normal tissue adjacent to the lesion was selected for use. Messenger RNA was prepared by chromatography on oligo (dT)-cellulose (Sambrook et al., 1989) and fractions containing polyadenylated RNA were pooled. First strand cDNA was synthesized as described (Ausubel et al., 1992). The ER-specific primer P4 (S-TGGGAGCCAGGGAGC-3’) was used to synthesize single-stranded cDNA from 5 pg of poly A+ RNA using AMV reverse transcriptase. P4 represents an antisense primer derived from the hER mRNA sequence immediately downstream of the SstI site at the 3’-end of the protein-coding region. 2.2. Polymerase chain reaction Products of the first strand cDNA synthesis reaction were used directly for PCR amplification as previously described (Wang and Miksicek, 1991). The primer pair Pl (S-AGGAGCTGGCGGAGG-3’) and P2 (S-GGTCAGTAAGCCCATCATCG-3’) was used to amplify the N-terminal portion of the ER protein-coding region, while P3 (5’-GCCCGCTCATGATCAAACGC-3’) and P4 (S-TGGGAGCCAGGGAGC-3’) were used to amplify the C-terminal portion (Fig. 1A). Thirty cycles of amplification were performed by denaturing 1 min at 95”C, annealing for 2 min at 45”C, and extending for 3 min at 70°C. Aliquots of the PCR products were analyzed by agarose gel electrophoresis and specificity of the reaction was confirmed by Southern hybridization (Southern, 1975) using a 32P-labeled synthetic hER mRNA as the probe. PCR amplification of genomic hER sequences utilized a similar protocol except that MgCl,was lowered from 2.5 to 1.5 mM. Aliquots (1 pg) of genomic DNA prepared according to Sambrook et al. (1989) from human lymphocytes or established cell lines were amplified with primers PS (5’-CCCGGGGCAGGGCCGGGG-3’) and H33 (5’-CGCGGCGTTGAACTCGTAG-3’) which lie within the first exon of the hER gene (Ponglikitmongkol et al., 1988). Thirty cycles of amplification were performed beginning with denaturation for 30 s at 96°C primer annealing for 60 s at 58°C and elongation for 60 s at 74°C. Taq DNA polymerase was added after the reaction temperature exceeded 85°C during the first cycle. Following cleavage with PvuII, genomic ampIifi~ation products were resolved on a native 5% polyac~lamide gel stained with ethidium bromide. 2.3. cDNA cloning and DNA sequence analysis Total products of the PCR amplification reactions were ligated into linearized pBS( + ) plasmid (Stratagene Cloning Systems), following digestion of both the

CellulmrEndocrinology 101 (1994) 101-110

PCR products and the cloning vehicle with Hind111 and Sst I. Bacterial transformants containing ER cDNA sequences were identified by colony hybridization (Sambrook et al., 1989) using appropriate 32P-labeled hER-specific RNA probes. Both single-stranded and double-stranded DNA were used for dideoxy sequencing (Chen and Seeburg, 1985) with primers covering the entire translated region of ER. Sequencing reactions were performed using a Sequenase II reagent kit and a-[35S]dATP according to the manufacturer’s instructions (United States Biochemical). 2.4. Construction of pER expression plasmids To construct ER expression vectors containing short 5’-open reading frame (S-ORF), the N-terminal pBS( + ) subclones were linearized Sst I (located at the N-terminal end of the cDNA), rendered blunt-ended with T4 DNA polymerase, and redigested with Hind111 (see Fig. 1A). To prepare the hER clones which lack the S-ORF, the PCR products were linearized with 7’thIIIl and repaired as above. N-terminal hER cDNA fragments containing either allele A or allele B (see below) were next shuttled through the polylinker region of pSK( + ) (pBluescript, Stratagene CIoning Systems) to place a unique BarnHI site upstream of the open reading frames. Restriction fragments were then excised with BumHI (5’) and Asp718 (3’) and ligated into BumHI/Asp718-cleaved pHCMti6TK BlueA” expression plasmid (see below). Complete hER cDNAs were reconstructed within these subclones by digesting the N-terminal intermediates with Hind111 and Sst I. These linearized vectors were ligated with HindIII/Sst I restriction fragments corresponding to the C-terminus of hER, prepared by excision from a PBS(+) C-terminal hER cDNA subclone. The resulting hER expression plasmids are denoted pERi through pER18. They each have a unique BumHI site at the 5’-end and unique SstI and EcoRI sites at the 3’-end of the cDNA inserts. These plasmids also contain T3 and T7 RNA polymerase promoters at the 5’- and 3’-ends of the cDNAs for preparing synthetic ER mRNA and anti-sense RNA probes, respectively. The parental vector pHCMv6TK BlueA+ is an expression vector derived from pBS( + ) which incorporates the transcriptional enhancer of human cytomegalovirus (HCMV), the Herpes simplex virus th~idine kinase (HSV tk) promoter, and polyadenylation signals from the SV40 T-antigen gene. This mammalian expression vector was designed for the facile insertion of cDNAs downstream of an active eukaryotic promoter by embedding the HSV tk restriction fragment inframe within the /3-galactosidase rr-peptide and retaining the multiple cloning site (Pstl to EcoRI) from pBS( + ). Accordingly, the ‘blue/white’ a-comple-

Y. Wang, R.J. Miksicek /Molecular

and Cellular Endocrinology

mentation assay can be used for the identification of recombinant expression clones within a suitable E. coli host strain. In addition, PHCMV~~TK BlueA + retains the high copy number colE1 replicon and the fl single-stranded origin of replication from pBS( + ) making this expression plasmid particularly useful for mutagenesis and DNA sequence analysis. 2.5. Cell culture and transfection HeLa cells were maintained in phenol-red free Dulbecco’s modified Eagle’s medium (DMEM) plus 10% ‘~Sst/ ERmRNA

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calf serum, supplemented with 5 mM Hepes (pH 7.4), 2 mM glutamine, 50 units/ml penicillin, and 50 pg/ml streptomycin. One day before transfection, the cells were transferred to DMEM containing 10% charcoaltreated calf serum. Cells were cotransfected with 1 pg of a PER expression plasmid and 16 pg of the estrogen-responsive reporter plasmid, pERE-TK-CAT (Klock et al., 1987) using the calcium phosphate precipitation method as described (Wang and Miksicek, 1991). Cell extraction and chloramphenicol acetyl transferase (CAT) assays were also performed as described using 100 pg of total cellular protein. CAT-specific activity PplldM 1

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Fig. 1. PCR amplification and cloning of hER cDNAs. (A) Diagram showing the location of primers for PCR amplification of hER cDNA and restriction sites for subcloning the amplification products into the pBS( + ) vector. Directions for transcription of T3 and T7 RNA polymerases are indicated by arrows flanking the multiple cloning site. This diagram also illustrates the structures of allele A and allele B, indicating the presence of the polymorphic PuuII site. (B) Mini-prep DNA from positive clones identified by colony hybridization was digested with PuuII and electrophoresed through a 1% agarose gel containing ethidium bromide. Lanes 1 through 23 represent individually isolated clones. The allele A and allele B isolates which were used for reconstruction of the respective intact hER cDNAs are represented by clone 6 (lane 6) and clone 5 (lane 5). In allele B, the extra PcuII site results in cleavage of the 601 bp fragment present in allele A to two DNA fragments of 320 bp and 281 bp.

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and Cellular Endocrinology 101 (1994) 101-110

was expressed as percent conversion of chloramphenico1 to its acetylated products.

3. Results

An incompletely studied question within the steroid receptor field asks to what extent structural variants of these receptors exist within the human population which may help to explain endocrinopathies or other disease states in which the steroid receptors may play a participatory role. Mutations and structural defects have been characterized within a number of the nuclear receptor family members that account for target organ resistance to androgens (McPhaul et al., 1993), thyroid hormones (McDermott and Ridgeway, 1993), and vitamin D (Wiese et al., 1993). Similarly, a number of variants of the estrogen receptor have been characterized in breast tumors (McGuire et al., 1991; Dotzlaw et al., 1992) or tumor cell lines (Graham et al., 1990; Wang and Miksicek, 1991) which are likely to contribute to alterations in the hormonal responsiveness of these cells. In order to pursue an analysis of naturally occurring variants of the estrogen receptor, we have devised a method for the efficient, directed cloning of ER cDNAs from human tissues using reverse transcription followed by PCR amplification (RT/PCR). Conditions for the directed cloning of hER cDNAs by RT/PCR were established using polyadenylated

RNA from normal human myometrium, a tissue which is known to express the estrogen receptor. The primers designed for both reverse transcription and PCR amplification were based on the previously reported sequence of the MCF7 ER cDNA (Green et al., 1986; Greene et al., 1986). Since the hER protein-coding sequence is 1785 nt in length, efforts to amplify the entire sequence using a single pair of primers were marginally successful. Therefore, we divided the coding region into two overlapping segments for separate amplification and subsequently rejoined the two fragments utilizing a common Hind111 site present in the overlap. The N-terminal segment was amplified with a pair of converging primers, PI and P2 (Fig. lA), corresponding to positions 1 to 15 and 1254 to 1273 of the mRNA sequence, respectively (numbering according to Green et al., 1986). Similarly, the primers selected for C-terminal amplification, P3 and P4, represent positions 1113 to 1132 and 2023 to 2037, respectively. An ER-specific first strand cDNA was generated by reverse transcription from primer P4 and was used directly for PCR amplification by Taq DNA polymerase. The amplified DNA products were confirmed to include ER-specific sequences by hybridization analysis (Southern, 1975) using a 32P-labeled antisense ER RNA probe (data not shown). The N- and C-terminal hER cDNA amplification products were then individually digested with SstI and Hind111 restriction endonucleases and ligated into the pBS( + ) plasmid (Stratagene). Recombinant plasmids confirmed by colony hybridization to contain hER cDNA sequences were selected for further analysis.

A

Fig. 2. PCR amplification of genomic DNA. (A) Diagram showing PCR amplification scheme of genomic DNA corresponding to exon I of the estrogen receptor gene for identification of the PvuII polymorphism. Primer pair P5 and H33 were used to amplify a DNA fragment of 349 bp. Presence of an internal PuuII site results in cleavage of this PCR product into two fragments of 221 bp and 128 bp. (B) PCR products were digested with P&I and analyzed on a 5% polyacrylamide gel. Lane 1 represents PCR reaction without genomic DNA added as a template; lanes 2 and 3, PCR products of two genomic DNA preparations from human myometrium; lanes 4 through 10, genomic DNAs isolated from peripheral blood samples from six healthy donors; lane 11, genomic DNA prepared from the breast cancer cell line MDA-MB231; lane 12, genomic DNA from the human uterine tumor cell line HeclA. Lane 3 corresponds to the myometrial tissue sample from which the hER cDNA clones described in this study were generated. Size markers (lane M) are pBS( + 1 plasmid cleaved with HinfI and R.saI.

Y. Wang, R.J. Miksicek /Molecular

and Cellular Endocrinology

3.2. Restriction mapping and revelation of a PvuII.p~lymorphism

Based on the MCF7 ER cDNA sequence (Green et al., 1986; Greene et al., 19861, several restriction endonucleases with unique (e.g. HindIII, BglII, XbaI) or multiple sites (e.g. PvuII, PstI) within the protein-coding region of the hER cDNA were chosen to confirm the restriction pattern of our clones. Most of the enzymes tested yielded digestion patterns as predicted from the MCF7 ER cDNA sequence. However, when PvuII was used to cleave the N-terminal hER cDNA clones, two distinctive patterns of digestion products were observed (Fig. 1B). In addition to the two PvuII sites present in the pBS( + ) vector, nine out of a total of 23 ER-positive clones (40%) have one internal PvuII site (designated allele A) while the remaining 14 clones (60%) have an extra PvuII site (allele B) that is not present in the hER cDNA isolated from MCF7 cells. Further analysis with a combination of other restriction enzymes indicated that this extra PuuII site is located near nucleotide position 300 of the hER mRNA (Fig. 1A).

Allele 6

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101 (195’4) 101-110

Following the discovery of this apparently polymorphic PuuII site, PCR amplification experiments were undertaken using genomic DNA as a template to determine whether it was also present in the hER gene. As indicated in Fig. 2A, a pair of primers (P5 and H33) was chosen which lie within the first exon of the hER gene (Ponglikitmongkol et al., 19881, flanking the presumed location of the novel PvuII site. Amplification products from this region, predicted to be 349 bp in size, were then cleaved with PvuII to assay for the presence of allele B. Results from the analysis of eleven genomic DNA samples (Fig. 2B) showed that the PvuII polymorphism was present only in the same uterine tissue sample from which the original cDNA’ was made (Fig. 2B, lane 3). Presence of the predicted 349 bp fragment in addition to two shorter fragments (221 and 128 bp) further indicated that this individual is heterozygous at this locus. Genomic DNA prepared from another uterine tissue sample (lane 21, DNAs obtained from blood samples of several healthy donors (lanes 4 to 10) and DNAs from four human tumor cell lines, MDA-MB231 (lane ll), HeclA (lane 12), HeLa, and T47D (data not shown) failed to show the presence

Allele A

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f

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Clone 1 G

A

T

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Fig. 3. cDNA sequences of selected regions of hER alleles A and B. (A) hER cDNA sequences in the vicinity of the polymorphic PuuII site within exon I. Shown is a sequencing autoradiogram indicating the presence of glutamic acid at amino acid position 22 of allele A and glutamine at the same position of allele B. In addition, there is a silent change within the codon for serine at amino acid position 10 in which allele A is encoded by TCT and allele B is encoded by TCC. (B) Sequence near amino acid 400 of hER. Sequencing results from three individually isolated ER cDNA clones showing that the amino acid in position 400 of wt-hER is a glycine residue encoded by GGG.

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and Cellular Endocrinology

of the B allele. Assuming that this genomic variant represents a true genetic polymorphism (as opposed to a somatic mutation), these data suggest that it is rare in the general population with a frequency of occurrence of 8% or less. 3.3. Sequence analysis of myometrial hER cDNA clones show differences compared to the ER cDNA from MCF7 cells Once the N- and C-terminal hER subclones were isolated, the dideoxy method (Chen and Seeburg, 1985) was used to sequence multiple clones in their entirety to confirm that sequence differences observed between these clones and the MCF7 receptor reflected authentic mRNA sequences from this tissue and were not due to misincorporation by Taq DNA.polymerase. We also sequenced multiple clones of allele A and allele B to identify any mutations that might come from PCR misincorporation. Using conservative estimates for the abundance of ER mRNA in the uterus (0.002% of total mRNA1 and the efficiency of reverse transcription (20%), it can be calculated that the starting amount (5 pg> of polyadenylated RNA used in our PCR amplification experiments corresponds to approximately 7 X lo6 molecules of hER cDNA. Therefore, even if misincorporation occurred during reverse transcription or during the first several rounds of PCR amplification, any unique error due to random misincorporation will rarely if ever exceed 1 part per million of the cloned products of the PCR reaction. Conversely, sequence

differences observed in a significant percentage of the PCR products must have been present at a similar frequency among the starting template molecules. In this study, we observed a number of random nucleotide changes with a frequency of occurrence of approximately 10p4, similar to the fidelity of Taq DNA polymerase reported in other studies (Dunning et al., 1988; Tindall and Kunkel, 1988). However, the sequence differences discussed below were consistent findings, compelling us to conclude that they reflect the characteristics of the starting RNA population. Sequence analysis of the N-terminal portion of the hER cDNAs confirmed the presence of a polymorphic PuuII site located at amino acid 22 which results in a change of Glu’* (GAG) to Gin** (CAG). The PuuII recognition sequence (CAGCTG) is thus created at nucleotide 298. This site agrees well with the PuuII site predicted to exist around nucleotide position 300 from the previous restriction analysis of minilysate DNA (Fig. 1) and genomic PCR amplification products (Fig. 2). Fig. 3A shows sequencing reactions performed on allele A and allele B near the polymorphic PuuII site. In addition to the G to C transversion at nucleotide 296, there is a silent change at Ser” that is linked to the PvuII polymorphism. In allele A (which is the same as the hER cDNA previously isolated from MCF7 cells) SerlO is encoded by TCT, while Set-” in allele B (containing the extra PvuII site) is encoded by TCC (Fig. 3A). The consistent pattern of segregation of these sequence differences among the multiple cDNAs analyzed argues further that they represent an authen-

DNA Allele

101 (I 994) 101-l 10

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A

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Allele B

s

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s

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CGC Genomic ER GOG T47D comparison of the myometrial cDNA clones with other known isolates of the hER cDNA. Differences are summarized between the nucleotide sequence of the wt-hER cDNAs reported in this study, the ER cDNA isolated from MCF7 cells (Green et al., 1986; Greene et al., 1986), and ER genomic DNA sequences (Ponglikitmongkol et al., 1988).

Fig. 4. Sequence

Y Wang, R.J. ~~~~ek/~olecular

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and Cellular Endocrinology I01 (1994) 101-110

man et al., 1988; Garcia et al., 19891, suggests that multiple alleles of the ER gene exist in the general population. Sequencing of multiple cDNA clones corresponding to the hER C-terminus revealed two additional sequence differences compared to the hER cDNA originally isolated from MCF7 cells. Shown in Fig. 3B are sequencing data near amino ,acid 400 from three independent clones isolated from human myometrium. These results demonstrate that at this site the sequence is represented by GGG encoding GUYED.Previous reports of the MCF7 cDNA sequence (Green et al., 1986; Greene et al., 1986) suggested that GTG encodes Val at position 400 of hER (Fig. 4). More recent analysis of hER DNA from a human lymphocyte genomic library (Ponglikitmongkol et al., 1988) noted the sequence as GGG (Gly‘?. It was later

tic genetic polymorphism rather than random artifacts of PCR amplification. It is interesting to note that the TCT (Ser”) and GAG (GAUGE)association in allele A is equivalent to the hER cDNA isolated from MCF7 cells. This association is also observed in the hER cDNA clones isolated from another human breast cancer cell line, T47D (Wang and Miksicek, 1991). However, a genomic hER clone sequenced in this region (Ponglikitmongkol et al., 1988) appears to represent a hybrid between the A and B alleles isolated in this study. in the genomic hER sequence, the TCC (Ser”) codon is found to be associated with GAG (GAUGE)which is different than either allele A (TCT with GAG) or allele B (TCC with CAG) (Fig. 4). This evidence, coupled with the identification of three additional restriction fragment length pol~o~hisms (RFLPs) (Castagnoli et al., 1987; Cole-

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Fig. 5. Transfection analysis of human estrogen receptor expression plasmids. (A) Map of the mammalian hER expression vector (PER) showing the locations of important functional elements. (B) Diagram showing the structure of hER cDNAs inserted into PHCMV~TK BlueA+. Myometrial hER cDNAs containing a 20 amino acid 5’-ORF are present in the expression clones labeled pER16 and pER17, while clones without this 5’-ORF are designated as pERi and pER19. As shown in the diagram, pER17 and pER19 also contain an additional PuuII site at amino acid position 22 which has been defined in this study as hER allele B. (C) Analysis of ho~one-dependent tran~riptional activation by the PER expression plasmids. Human ER cDNA expression plasmids were cotransfected into HeL.a cells with the estrogen-responsive reporter plasmid pERE-TK-CAT to assess their ability to activate transcription in vivo in response to hormone. The control samples refer to transfections performed with the parental expression vector pHCMV?K BlueA+ which lacks an ER cDNA insert. ‘ - / + ’ indicates the absence or presence of 5 nM 17&estradiol during the 48 h expression period following transfection. Numbers at the bottom of the figure indicate the percentage of [t4C]chloramphenicol converted to its acetylated forms by the CAT enzyme.

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determined that the sequence difference in MCF7 cells was probably due to a cloning error as determined by PCR amplification and RNase protection analysis of mRNA from MCF7 cells (Tora et al., 1989b). Glycine at this position is also found to be conserved in ER cDNAs isolated from five other species (Krust et al., 1986; White et al., 1987; Weiler et al., 1987; Koike et al., 1987; Pakdel et al., 1990). This observation is of significance because the Gly400 to Va1400mutation has been found to decrease the stabiiity of this protein and to affect hormone binding by the receptor expressed from this cDNA above 25°C (Tora et al., 1989b). A final variation in the sequence of hER is present at Thr594. As shown in Fig. 4, hER cDNAs isolated from the uterine tissue and from genomic DNA have an ACG codon at this position while hER cDNA from MCF7 cells has a silent mutation in which threonine is encoded by ACA. Since this variant (ACA, Thr594) has not been observed in other hER cDNA clones isolated to date, it may represent an additional cloning artifact present within the original MCF7 cDNA clone or an independent sequence polymorphism. 3.4. Functional analysis of wt-hER cDNAs From the design of our RT/PCR strategy, we would predict that amplification of the N-terminus of hER using primers Pl and P2 would result in the inclusion of a 5’-short open reading frame (5’-ORF) of 20 amino acids located 50 nucleotides upstream of the major open reading frame of the hER protein (Green et al., 1986; Greene et al., 1986). All of the N-terminal cDNA clones that we have identified have this short open reading frame and the sequence of this region is identical to that of the MCF7 ER cDNA. Although the presence of this short 5’-ORF was noted at the time the hER cDNA was originally cloned and sequenced, it appears that no attempt has been made to determine whether its presence affects the expression or the function of the adjacent estrogen receptor reading frame. Various cDNA clones differing at their N-termini (Fig. 5A) were introduced into the multiple cloning site of the mammalian expression vector pHCMti”TK BlueA+ to generate a series of PER expression clones (Fig. 5A). Allele A is present in pER16 and pER18, while clones which have the additional PvuII site (allele B) are named pER17 and pER19. The pHCMp6TK BlueA+ derivatives which include the 5’-ORF are labeled as pER16 and pER17 (Fig. 5B). As with other steroid receptors, hER acts as a transcription factor capable of stimulating gene expression in response to its principal physiological ligand (17/3-estradiol). This receptor binds to one or more estrogen responsive elements (ERE) in the control region of estrogen-regulated genes, such as the vitellogenin genes of Xenopus faeL+s (Klein-Hitpass et al.,

1986). Through poorly characterized interactions with other transcription factors, hER regulates the initiation of mRNA synthesis by RNA polymerase II. To verify the transcriptional activity of the newly cloned myometrial ER cDNAs in vivo, we cotransfected HeLa cells with the hER expression plasmids (pER16 through pER18) and an estrogen-responsive reporter plasmid (pERE-TK-CAT) &lock et al., 1987). As shown in Fig. 5C, induction of pERE-TK-CAT in response to 5 nM estradiol requires the presence of cotransfected estrogen receptor, as the pHCMp6TK BlueA+ cloning vector without an ER cDNA fails to elicit an increase in CAT enzyme expression (lanes 1 and 2). However, when an hER cDNA is transfected into HeLa cells together with the reporter plasmid (lanes 3 through lo), CAT activity is significantly increased (12- to 15-fold) in the presence of estradiol. The basal activity of the reporter plasmid is slightly higher in the presence of cotransfected receptor than in its absence. This is likely to be due to the presence of low amounts of steroids that remain in the charcoaltreated serum used for cell culture. It is apparent from Fig. 5C that both alleles of hER activate transcription to approximately the same extent (compare pER16 with pER17, and pER18 with pER19). The presence of the 5’-short ORF in the ER expression plasmids pER16 and pER17 (which give approximately 35% substrate conversion) seems to produce only a modest reduction in CAT activity compared to pER18 and pER19, both of which lack the 5’-ORF (and give approximately 45% substrate conversion in the presence of estradioll. The lower activities of pER16 and pER17 could result from competition of the short 5’-ORF for ribosome binding to the major open reading frame of the ER message, causing impaired translation and slightly reduced levels of ER protein. This explanation is also consistent with the higher basal activities seen with pER18 and pER19, versus pER16 and pER17 since the activity of pERETK-CAT in the absence of estradiol tends to correlate with the efficiency of ER expression (Wang and Miksicek, unpublished observations). However, these differences are relatively small and lead us to conclude that the overall transcriptional activities of pER16, pER17, pER18 and pER19 are comparable and that the 5’-ORF plays little, if any, effect in regulating translation of the hER message, at least in the context of our experimental design.

4. Discussion Prior to this study, a cDNA corresponding to the hER mRNA was isolated from the human breast cancer cell line MCF7 (Walter et al., 1985). Since this cDNA was obtained from a tumor source, it was not

Y. Wang, R.J. Miksicek / Molecular and Cellular Endocrinology 101 (1994) 101-110

entirely clear whether this clone would be equivalentto a wild-type hER cDNA derived from normal human tissue. The present study was undertaken to address this important question and to establish the wild-type sequence off an hER cDNA isolated from non-diseased human tissue. Using reverse transcription and PCR amplification for directed cDNA cloning, we have successfully isolated two closely related hER cDNAs from human myometrium and determined the complete nucleotide sequence of their protein-coding regions. All of the cDNAs which we have characterized from normal uterine tissue are predicted to encode fully intact receptors, in contrast to studies using similar techniques to analyze ER cDNAs from breast tumors and tumor cell lines (McGuire et al., 1991; Wang and Miksicek, 1991). This indicates that ER mRNA variants derived by ‘exon skipping’ do not appear to occur in the normal human uterus. The cloning revealed the presence of a PLIUII polymorphism within the N-terminal region of the hER cDNA. The existence of this PrluII polymorphism has been independently confirmed by PCR amplification of genomic DNA prepared from the same tissue. Analysis of thirteen additional genomic DNA samples prepared from tissues, cell lines, and blood samples indicates that the polymorphic P~juI1 site which we identified in exon I is so far unique to the specimen from which it was isolated. A much larger number of human DNA samples will be required to confirm that this is an inherited polymorphism rather than an isolated somatic mutation and to accurately determine its frequency within the human population. Previous studies of polymorphic restriction sites within the human ER locus have identified RFLPs for BbuI (Garcia et al., 19891, &I (Coleman et al., 19881, and Pr~uI1 (Castagnoli et al., 1987). A detailed study of genomic DNAs prepared from various human breast cancer cell lines as well as peripheral blood samples was conducted by Hill et al. (1989). They suggested that this PvuII polymorphism was located near the DNA- and hormone-binding domains. Using the PCR technique, Yaich et al. (1992) studied the exon structure of the ER gene in 26 primary breast cancers (containing both ER-positive and ER-negative specimens) and found no major deletions or rearrangements within the ER gene. They further identified the polymorphic PvuII site described by Castagnoli et al. (1987) as lying within the first intron, immediately upstream of exon II. Subsequent screening of 257 primary breast cancers and 140 peripheral blood DNAs suggested that this polymorphism is not associated with ER content or patient age at tumor diagnosis as was previously suggested (Hill et al., 1989). Our results indicate that the PuuII polymorphism which we identified within the protein-coding region of the ER cDNA (exon 1) is different from that previously reported. Since the hER

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cDNA representing allele B (containing the extra PuuII site) harbors a change in amino acid sequence of the encoded receptor and has the potential of affecting protein function, transfection experiments were conducted to characterize the behavior of these cDNA clones. Both alleles are fully functional with respect to hormone-dependent activation of transcription (Fig. 50 and are indistinguishable with respect to their DNA-binding and ligand-binding properties (data not shown). Previous cloning of hER cDNAs from the human breast cancer cell line MCF7 indicated the existence of a cloning artifact which resulted in the change of Gly 400 to Va1400 (Tora et al., 1989b). We have confirmed that the wild type hER cDNA encodes a glycine at amino acid position 400 by RT/PCR amplification of mRNA isolated from normal human myometrium. Although there have been reports that wt-hER with acGly 4oo results in constitutive, estrogen-independent tivation of transcription (Tora et al., 1989b; Tzukerman et al., 19891, our data indicate that the appearance of constitutive activation might be due to the presence of trace amounts of steroid that remain in the charcoaltreated serum used for cell culture. Our experience shows that the non-mutated hER is capable of inducing the expression of a reporter plasmid lo- to 15-fold in response to low concentrations of 17&estradiol (see Fig. 50 It should be noted that allele A of hER which we isolated from human uterine tissue is identical in sequence to the hER cDNA found in T47D cells (Wang and Miksicek, 1991) and to the N-terminal portion of the hER cDNA isolated from MCF7 cells (Green et al., 1986; Greene et al., 1986). These results, together with the occurrence of at least three additional RFLPs (Castagnoli et al., 1987; Coleman et al., 1988; Garcia et al., 19891, indicate that a minimum of four alleles of the ER gene exist in the human gene pool. From these studies, one can speculate that additional estrogen receptor alleles will be identified in the future. An open question remains whether any correlation exists between individual alleles of the estrogen receptor and pathological conditions such as a high frequency of spontaneous abortion, impaired fertility, developmental abnormalities, and cancers of the reproductive tract. Using this method we have identified three variant hER mRNAs present in the breast cancer cell line T47D which harbor deletions of internal exons of the hER gene (Wang and Miksicek, 1991). The presence of these mRNA variants in uncloned RNA was independently confirmed using RNase protection assays (Miksicek et al., 19931, demonstrating that they are not an artifact resulting from the use of PCR amplification for directed cDNA cloning. Through this study we have established a convenient method that can be readily employed to investigate mutations within the hER

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and Cellular Endocrinology 101 (I 994) 101 -I 10

mRNA in human tissues and tumor specimens. Advances in DNA sequence analysis of PCR-amplified DNA which avoid the necessity of prior cloning will simplify the task of identifying possible defects in hER that might be associated with reproductive tumors or other disease states.

5. Acknowledgements

We would like to thank P. Chambon and colleagues for providing HE0 as well as G. Schiitz and colleagues for the plasmid pERE-TK-CAT. This work was supported by grant CD-479 from the American Cancer Society and grant CA47384 from the U.S.P.H.S. to R.M.

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