Differentially Expressed Messenger RNA Isoforms of the Human Estrogen Receptor-{alpha} Gene Are Generated by Alternative Splicing and Promoter Usage

Gilles Flouriot, Caroline Griffin, Maryrose Kenealy, Vera Sonntag-Buck and Frank Gannon

European Molecular Biology Laboratory (G.F., V.S-B., F.G.) D-69117, Heidelberg, Germany
National Diagnostic Centre (C.G., M.K.) University College Galway, Ireland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The isolation and characterization of several new human estrogen receptor-{alpha} (hER{alpha}) mRNAs are described. Together with those previously identified, they give rise to a total of six hER{alpha} mRNA isoforms (A–F hER{alpha} mRNAs). Produced from a single hER{alpha} gene by multiple promoter usage, all these transcripts encode a common protein but differ in their 5'-untranslated region as a consequence of alternative splicing of five upstream exons (1B–1F). RT-PCR and S1 nuclease mapping analysis of these different hER{alpha} mRNA isoforms revealed a differential pattern of expression of the hER{alpha} gene in human tissues and cell types. The A hER{alpha} mRNA is the main isoform detected in mammary glands or in the tumor cell lines derived from this tissue. In endometrium, the predominant forms are the A and C hER{alpha} mRNA isoforms, whereas the C and F hER{alpha} mRNA isoforms are the major forms detected in ovary. Finally, high levels of the E hER{alpha} mRNA isoform are restricted to the liver with an increased expression in females. Taken together, our results demonstrate that the hER{alpha} gene is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estradiol (E2), a steroid hormone principally produced in ovaries, is involved in the regulation of a wide range of physiological processes (1, 2, 3). In addition, its association with pathological events such as breast and endometrial cancer has been well demonstrated (4). The physiological changes induced by E2 often result from modifications in the expression patterns of specific target genes and are mediated by intracellular receptors. To date, two nuclear estrogen receptors (ER{alpha} and -ß), coded by different genes, have been identified (5, 6, 7). These two receptors belong to the superfamily of steroid/thyroid hormone/retinoic acid receptors whose members act as ligand-inducible transcriptional factors (8, 9, 10). These factors are structurally organized into six defined domains A–F, which control functions such as DNA and ligand binding (C and E, respectively) and gene transactivation (A/B and E) (8, 9, 10). As has been shown more recently for ERß (6, 7, 11), the ER{alpha} is expressed in a great variety of cell types and tissues, in agreement with the diversity of physiological effects of its ligand. In addition to its presence in reproductive tissues such as ovary, uterus, or mammary gland (12, 13, 14), ER{alpha} expression was also detected in bone (15, 16), fat, and vascular tissues (17, 18), liver (13), skeletal muscles (19), pituitary gland, and central nervous system (13, 20, 21). The expression level varies considerably among these tissues as well as within a single tissue at different physiological stages (13, 22, 23). Moreover, ER{alpha} expression has been shown to be specifically stimulated or repressed by the ligand itself and other hormones in a tissue-dependent manner (12, 13, 24, 25). The differential and spatio-temporal expression of ER is most probably responsible for the specificity and diversity of the effects of E2. Thus, the elucidation of the molecular mechanisms controlling the tissue-specific pattern of ER{alpha} should provide a starting point for understanding how the pleiotropic effects of its ligand are integrated into a wide range of biological processes.

Previous studies on the structure and organization of the human ER{alpha} (hER{alpha}) gene indicated that the transcriptional activity of this gene was probably controlled by more than one promoter (23, 26, 27). We reported that a portion of the 5'-flanking region of the ER{alpha} gene, at approximately -1.9 kb upstream from the formerly assigned transcription start site (5), presented a high homology with the 5'-end of the rat ER{alpha} cDNA and exon 1 of the mouse ER{alpha} gene (26). This predicted the existence of a second hER{alpha} mRNA, which would arise from splicing of an upstream exon to the 5'-untranslated region (UTR) of the previously designated exon 1. Such a transcript was demonstrated by RT-PCR (26). Further investigation into the expression of these two hER{alpha} mRNAs suggested the likely existence of additional hER{alpha} transcripts. Using the rapid amplification of cDNA ends (RACE) methodology, we have now identified four new hER{alpha} mRNAs,1 which together with those previously characterized, give rise to a total of six hER{alpha} mRNA isoforms (A–F hER{alpha} mRNAs). All of these transcripts are generated by differential promoter usage and differ in their 5'-UTR because of an alternative splicing event. Furthermore, these transcripts are differentially expressed in human tissues or cell types.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Evidence for Additional hER{alpha} mRNA Isoforms
It has been previously shown that the hER{alpha} gene is transcribed from at least two promoters, which are separated by approximately 2 kb (26). The two transcripts, called A and C (for reasons that will be explained below), differ in their 5'-UTR, but are almost identical in size due to the fact that transcript C, initiated at the upstream exon (1C), splices into exon 1A at position +163 (Fig. 1AGo). To determine precisely the 5'-extremities of these two hER{alpha} mRNAs and to quantify their relative abundance, especially in the breast carcinoma cell line MCF7, which expresses relatively high levels of ER{alpha}, S1 nuclease mapping and primer extension experiments were performed.



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Figure 1. Determination of A and C hER{alpha} mRNA Transcription Start Sites by S1 Nuclease Mapping and Primer Extension and Identification of the Alternative Splicing Event

A, Schematic representation of exons 1A and 1C of the hER{alpha} gene and the corresponding transcripts. The transcription start sites of the hER{alpha} gene are represented by broken arrows. The initiation codon ATG and the splicing site position in exon 1A (+163) are indicated, and the two single-stranded S1 probes A and C specific for the hER{alpha} transcripts A and C, respectively, as well as primer {Sigma}, are shown. Also shown are the approximate locations of the primers used to prepare the S1 probes and primer {Sigma}. B, Thirty micrograms of total RNA from MCF7 cells and 30 µg of tRNA (as a negative control) were hybridized to the labeled S1 probes A and C and treated with S1 nuclease, and the resistant hybrids were separated on a sequencing gel as described in Materials and Methods. The undigested probe is shown in a separate lane. The corresponding DNA sequences prepared with the same primers as used for probe synthesis and run in parallel on the same gel are shown. The sequences shown on the left correspond to the coding strand. The transcription start sites and the splice site position are indicated. C, Thirty micrograms of total RNA from MCF7 and endometrium as well as 30 µg of tRNA (as a negative control) were hybridized to the primer {Sigma} and treated with the reverse transcriptase, and the extension products were separated on a sequencing gel as described in Materials and Methods. The transcription start sites of A and C hER{alpha} mRNAs and primer {Sigma} position are indicated.

 
Because of the weak expression level of hER{alpha} mRNAs in most cells and tissues, we developed sensitive S1 nuclease mapping and primer extension methods. They involve the use of biotinylated single-stranded DNA templates bound to streptavidin-coated magnetic beads to prepare highly labeled single-stranded DNA probes or long primers by extension from a specific primer (28, 29). Using the new technique, two single-stranded DNA S1 probes A and C, specific for the corresponding hER{alpha} mRNAs, were prepared from genomic and RT-PCR products, respectively (see Materials and Methods). These two probes shared a common region 3' to the splice site position (Fig. 1AGo). After probe A hybridization with total RNA from the MCF7 cell line and S1 nuclease digestion, four major protected fragments were detected, with their 5'-ends located at 70, 229, 232, and 252 nucleotides upstream from the start of the hER{alpha} open reading frame (ORF) (Fig. 1BGo). No protected fragments were seen with tRNA used as a negative control. The smallest fragment corresponded to hER{alpha} mRNAs that remained homologous to probe A as far as the splice site position at +163 (26) and then diverged in their 5'-ends from probe A complementary sequences. The three other sites confirmed the transcription start site of transcript A already characterized by Green et al. (+1) (5) and identified two new ones (-20, +4). These sites have also been mapped in primer extension experiments using either primer A specific to A hER{alpha} mRNA (data not show) or a primer {Sigma} complementary to common sequences of A and C hER{alpha} mRNAs (Fig. 1CGo). The relative expression level detected at these three start sites suggested that approximately half of the total hER{alpha} mRNA expression level in MCF7 is due to transcript A. The other half, detected at the splice site position in the S1 nuclease mapping experiment, should correspond to transcript C if the hER{alpha} gene is transcribed only from the two promoters (A and C) previously described (Fig. 1AGo) (26).

In the corresponding experiment with a probe specific for transcript C, two protected fragments, in addition to the expected protected fragment detected at the splice site position (+163), were observed (Fig. 1BGo). Taking into account the intronic region -1860 to +162 (previously described in Ref. 26), the sites identified by the 5'-ends of the two longest fragments are located at 1974 and 2000 bp upstream from the transcription start site characterized by Green et al. (5). The same two sites were mapped by a primer extension analysis using either primer {Sigma} (Fig. 1CGo) or a specific primer for transcript C (data not shown), thus confirming that they correspond to the transcription start sites of C hER{alpha} mRNA isoform. It should be noted that the expression level in MCF7 of the hER{alpha} mRNAs containing exon 1C sequences (<10% of the total hER{alpha} mRNA level), was nevertheless much lower than suggested by a two transcript explanation of the S1 nuclease analysis of the hER{alpha} mRNAs/probe A hybrid results where approximately 50% of hER{alpha} transcripts diverged from A hER{alpha} mRNA at the splice site position. Moreover, the proportion of hER transcripts detected by probe C at the splice site position (at least 90% of the total hER mRNA level in MCF7) was also more than the 50% predicted from transcripts A’ expression level. These results suggested the existence of additional hER{alpha} mRNA isoforms in MCF7 obtained by the splicing of unidentified upstream exon(s) to the acceptor splice site of exon 1A at position +163. This hypothesis was strengthened by a primer extension experiment using the primer {Sigma}, which was designed to hybridize hER{alpha} mRNAs in a region downstream of the splice site position and was thus able to be extended to all the 5'-extremities of hER{alpha} transcripts. Results showed indeed several other extension products in addition to those related to A and C hER{alpha} mRNAs (Fig. 1CGo). These products might arise from new hER{alpha} transcripts and partial extension on hER{alpha} mRNAs. Interestingly, these results suggested also that the hER{alpha} gene exhibits alternative splicing in a tissue-specific manner since the primer extension pattern obtained from MCF7 was different from that detected in endometrium, especially for the C hER{alpha} mRNA isoform whose expression was relatively higher in endometrium than in MCF7.

In conclusion, these data clearly indicated the existence of more than two hER{alpha} mRNA isoforms whose expression level may be controled in a tissue-specific manner.

Four Novel hER{alpha} mRNA Isoforms: B, D, E, and F hER{alpha} mRNAs
To amplify new 5' mRNA extremities of the hER{alpha} gene, a variation of the inverse PCR technique was performed (30). The 5'-ends of three additional hER{alpha} mRNA isoforms (B, D, and F hER mRNA isoforms) were thus cloned from MCF7 total RNA, together with the two previously described (A and C hER{alpha} mRNA isoforms) (Fig. 2Go). In agreement with the S1 nuclease analysis of the hER{alpha} mRNAs/probe A hybrids, the most frequent cloned isoform was A hER{alpha} mRNA. Clones containing B, C, D, and F mRNA sequences were much less frequent (~8–10% for each type). All of the 5' hER{alpha} cDNA ends contained common exon 1A sequences up to the splice site position that is located 5' to the translational initiation codon of the hER{alpha} gene. All the cDNAs diverged from each other, however, immediately upstream from this position. Due to the RACE technique used to generate the 5'-terminal hER{alpha} cDNA regions (nick translational replacement of the mRNA was used to synthesize the second strand cDNA), the sequences of the new B, D, and F hER{alpha} mRNAs are probably incomplete at their 5'-ends. This possibility is supported by the fact that clones containing A and C mRNA sequences were deleted of some nucleotides at the 5'-end of these transcripts.



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Figure 2. Partial DNA Sequences of B, D, E, and F hER{alpha} cDNA Isoforms and Their 5'-Flanking Genomic Regions

||—• and {circ}—|| mark the 5'-end of the RACE product sequences and the 3'-end of sequences obtained by the genomic walking technique, respectively. Putative TATA and CAAT box as well as AP1 and half-estrogen responsive element (1/2 ERE) sequence motifs are underlined and annotated. The transcription start sites are indicated by broken arrows. The open triangles show exon junctions. The sORFs are underlined. The first amino acids of the ER{alpha} protein are indicated below the corresponding hER{alpha} cDNA sequences.

 
The existence of a further variant at the 5'-end was suggested by S1 nuclease mapping experiments on liver total RNA using a probe specific for F hER{alpha} mRNA isoform (see later sections on RNA analysis) (Fig. 5BGo). The new hER{alpha} transcript (E hER{alpha} mRNA isoform) shared common sequences with F hER{alpha} mRNA isoform as far as 131 nucleotides upstream from the splice site position in exon 1A and then diverged from this position to give different 5'-end sequences. Recently, the identification of a new 5'-hER{alpha} cDNA end, which corresponds to the E hER{alpha} mRNA isoform, was reported (31).



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Figure 5. hER{alpha} mRNA Isoform Distribution Analysis by S1 Nuclease Mapping

A, Experimental design for hER{alpha} mRNAs detection, indicating the location and the size of each single-stranded probe (A'–F') and each protected fragment obtained after S1 digestion of the probe/hER{alpha} mRNA hybrids. Each probe was specific for one of the hER{alpha} transcripts (e.g. A hER mRNA) but was also able to partially protect the other hER{alpha} mRNA isoforms [e.g. ({Sigma}-A) hER mRNA] up to the splice site position(s). Note that probes E' and F' yielded an additional protected fragment since E and F hER{alpha} transcripts also shared, in addition to the common part of exon 1A, one part of exon 1E. The probes were designed to contain vector sequence in their extremity (denoted by the thinner black line) to discriminate between undigested probes and specific protected fragments (see Materials and Methods for the preparation of the probes). B, S1 nuclease mapping assays of the hER{alpha} mRNA isoforms were performed as described in Materials and Methods, with the single-stranded probes A'–F' specific for each hER{alpha} transcript (see above) and using 30 µg of total RNA from various sources as indicated at the top of each lane. Yeast total RNA was used as a negative control. >> Indicates nonspecific protected fragment. Integrity of the different RNA samples was checked by performing a S1 nuclease protection assay on ubiquitin mRNA. Tissues from Caucasian males are indicated by *.

 
Therefore, the hER{alpha} gene is transcribed in at least six different transcripts that differ in their 5'-UTR as a consequence of the alternative splicing of several upstream exons.

Proximal Promoters of the B, D, E, and F hER{alpha} mRNA Isoforms and Preliminary Organization of the 5'-Region of the hER{alpha} Gene
The evidence of previouly unidentified upstream exons that were alternatively spliced to the acceptor site of exon 1A (at position +163) prompted us to further investigate the 5'-genomic organization of the hER{alpha} gene.

The specific 5'-cDNA end sequences of B hER{alpha} mRNA isoform were totally homologous to a genomic region located between exon 1A and 1C that had previously been sequenced (32). This genomic region was therefore part of a new exon whose 3'-end was positioned at 168 nucleotides upstream from the transcription start site characterized by Green et al. (5). This new exon is referred to as exon 1B (Figs. 2Go and 3Go). Several transcription start sites of B hER{alpha} mRNA were determined by S1 nuclease protection analysis using probe B spanning the region -150 to +283 (numbering from one of the main start sites of B hER{alpha} mRNA isoform) (Fig. 2Go) and further verified by a primer extension experiment with primer B (data not shown).



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Figure 3. Preliminary Schematic Representation of the 5'-Region of the hER{alpha} Gene

The exons 1A–1F are indicated with black rectangles, and their relative order in the hER{alpha} gene is shown. The approximate location of the primers used for the RACE and the genomic walking are shown by short arrows.

 
The 5'-cDNA end sequences specific to D and E/F hER{alpha} mRNA isoforms were not found in the 3.2-kb genomic region upstream from exon 1A that had been previously analyzed (32). To attempt to locate the related exons, a PCR analysis was performed by two rounds of 30-cycle PCR amplification on human genomic DNA. The 5'-primers and nested primers used for the genomic amplification were specific to the new 5'-cDNA end sequences [(D1, D2) and (E/F1, E/F2) for D and E/F hER{alpha} mRNA isoforms, respectively] with the 3' and nested primers (C4 and C5) from the 5'-end of the known genomic sequences of the hER{alpha} gene (27, 32) (see Fig. 3Go for primer location). A specific PCR-amplified genomic fragment of approximately 620 bp was obtained, which allowed one of the new exons, exon 1D, to be positioned at approximately 3.7 kb upstream from the transcription start site characterized by Green et al. (5) (Fig. 3Go). The amplification of the genomic region separating exon 1E (exon shared by isoforms E and F) from exon 1D was unsuccessful. It was also impossible to amplify from human genomic DNA a specific PCR product between isoform F-specific sequences and exon 1E. This indicated that these two regions were encoded by different exons separated by an intron that was sufficiently long to preclude amplification.

Using a rapid genomic walking technique (see Materials and Methods), the 5'-flanking regions of the 5'-RACE products obtained for D and F hER{alpha} isoforms, as well as the 5'-flanking region of the exon shared by isoforms E and F (exon 1E), were amplified. The sizes of these genomic PCR products were approximately 430, 400, and 170 bp, respectively. Figure 2Go shows the hER{alpha} 5'-untranslated and flanking sequences obtained from these PCR products. The 5'-flanking sequences of exon 1E were identical to the sequences of the 5'-hER{alpha} cDNA end recently identified (31). Therefore, this result demonstrated that the 5'-end of isoform E was encoded by only one exon (exon 1E), whereas that of isoform F contained two exons of which the upstream one (exon 1F) is spliced to an acceptor site located in exon 1E (Fig. 2Go). The sequences adjacent to this site in exon 1E correspond to the splicing consensus acceptor sequences (AG).

Using MCF7 and liver RNA, the localization of the transcription start sites of E and F hER{alpha} mRNA isoforms was determined both by S1 nuclease protection analysis using the probes E and F spanning the corresponding 5'-untranslated and cloned flanking sequences (see Materials and Methods for the probe preparation) and by primer extension with the long primer E/F (Fig. 2Go and data not shown). The 5'-extremity of D hER{alpha} mRNA isoforms was only detected by primer extension using a long primer (primer D). S1 nuclease mapping experiment specifically designed to map the 5'-end of D isoform failed to detect any signal.

As previous studies suggested the existence of a further transcription start site upstream of exons 1C at position -3090 (27), S1 nuclease experiments on MCF7 RNA were performed using probes spanning the region -2500 to -3250. No transcription start sites were detected and the probes were fully protected. As this result could be due to residual DNA fragments, an RT-PCR experiment using a primer 5' to position -3090 (primer C6) and a primer in exon 1A (primer VI) was performed (see Fig. 3Go for primer location). An RT-PCR product, the size of which corroborated with a splicing event between exons 1C and 1A, was obtained indicating that the genomic region located upstream of -3090 (i.e. between exons 1C and 1D) could also be transcribed and spliced to the body of the hER gene. This could be due to some of the pre-D hER{alpha} mRNAs that were not spliced as expected using the normal splicing donor sequence of exons 1D, but by utilizing those of exons 1C. Similar observations were obtained for the genomic region located between exons 1C and 1B (data not shown), but the S1 nuclease and primer extension experiments demonstrated clearly that both C and B isoforms existed independently of such read through.

Taken together, all of these results demonstrated that the hER{alpha} gene is a complex genomic unit controlled by at least six promoters (A–F) and exhibiting alternative splicing of five upstream exons (1B–1F).

Differential Expression of the hER{alpha} mRNA Isoforms
To get an overview of the pattern of expression of the hER{alpha} mRNA isoforms in various human tissues and cell lines, an RT-PCR analysis was performed. Single-stranded cDNAs were synthesized from total RNA of various sources using a hER{alpha} gene-specific primer (I) chosen from the 3'-UTR region of the hER{alpha} gene (exon 8) (Fig. 4AGo). The different hER{alpha} cDNAs were amplified by two rounds of PCR (30 cycles each) utilizing a common 3'-primer (II) and nested primer (III) located upstream from primer I in exon 8, in combination with 5'-primers and nested primers specific for the different hER{alpha} mRNA 5'-extremities (Fig. 4AGo). Thus, approximately all the coding region of the hER{alpha} transcripts was amplified. Results from this study showed a differential expression of hER{alpha} mRNA isoforms among the tested samples (Fig. 4BGo). The D hER{alpha} mRNA isoform displayed a more restricted expression pattern than the A, B, and C hER{alpha} mRNA isoforms, whereas the E and F hER{alpha} mRNA were the only forms detected in most tissues and cell lines tested. HeLa, which is usually considered to be an ER-negative cell line, expressed PCR-detectable amounts of the isoforms B, E, and F. It should also be noted that isoforms E and F were the only hER{alpha} transcripts detected by RT-PCR in the two osteoblast cell lines, HOS TE 85 and SAOS.



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Figure 4. RT-PCR Analysis of hER{alpha} mRNA Isoforms

A, Schematic representation of the experimental design for the RT-PCR analysis. Open boxes indicate the unique (1A–1F) and common (1 2 3 4 5 6 7 8 ) exons encoding each hER{alpha} mRNA isoform. Approximate locations of primers are shown by short arrows. Primer I, located in the 3'-UTR of exon 8, was used to prime hER{alpha} cDNA synthesis by reverse transcriptase. Primers A1–F1, which are specific for each hER{alpha} cDNA 5'-region, were used in a first round of PCR amplification with primer II, which is nested to primer I in exon 8. A second round of PCR reaction was then performed with specific (A2–F2) and common (III) nested primers. The oligonucleotide probe (ex. 2) from exon 2, common for all the isoforms, was used to confirm the specificity of the PCR products. B, The hER{alpha} cDNA isoforms were amplified as described above, using total RNA from various sources as indicated at the top of each lane. Yeast total RNA was used as a negative control. PCR products were electrophoresed through an agarose gel and transferred by Southern blot to a membrane that was then hybridized with the oligonucleotide probe ex. 2 as described in Materials and Methods. Positions of migration of the molecular size markers for each gel are shown on the left side of the figure. Tissues from Caucasian males are indicated by *.

 
In addition, this study showed also that the size of the amplified cDNAs was as expected and after Southern blotting, the hybridization of these PCR products with various oligonucleotide probes recognizing specifically the different eight coding exons of A hER{alpha} mRNA isoform (the only hER{alpha} mRNA fully characterized) demonstrated that sequences encoding the region A–F were present in all ER{alpha} mRNA isoforms (Fig. 4BGo shows only the results obtained with the oligonucleotide probe ex. 2). hER{alpha} cDNAs with a deletion of exon 7 were also observed in the tissues and cell lines in which the normal product was detected (data not shown). This deletion induces a premature stop in the hER{alpha} encoding ORF. Finally, the B hER{alpha} mRNA isoform detected in liver was longer than expected due to the presence of the intronic sequences located between exons 1B and 1A.

To estimate quantitatively the relative abundance of the different hER{alpha} mRNA isoforms in the various RNA samples, S1 nuclease mapping experiments were performed using single-stranded DNA probes specific for each hER{alpha} transcript (probes A', B', C', D', E', and F') (Fig. 5AGo). Due to the common sequence 3' to the splice site position, these probes were also able to measure the residual expression resulting from the sum of the expression of the other isoforms [for example ({Sigma} - A) hER{alpha} mRNA in Fig. 5AGo]. To distinguish between undigested probes and specific protected fragments, all of the probes contained additional sequences in their 3'-ends that originated in the vectors used for the single-stranded probe preparation. Finally, the integrity of the RNA from the different tissues and cell lines tested was checked by performing a S1 nuclease protection assay on ubiquitin mRNA, a housekeeping messenger. It should be noted that, as for other housekeeping messengers, ubiquitin mRNA may present some tissue variation in its expression level, and it is generally more highly expressed in cultured cells than in tissues (33, 34). As shown by the results in Fig. 5BGo and also their quantification summarized in Table 1Go (where the data are expressed as percentage of the total hER{alpha} mRNA expression detected in MCF7 cells), there was tissue specificity in the level of expression of the different hER{alpha} mRNA isoforms. Briefly, transcript A was the major form (~50%) expressed in the mammary gland or the cell lines derived from this tissue (MCF7, T47D, and ZR 75–1). In endometrium, the predominantly expressed forms were the A and C hER{alpha} mRNA isoforms, whereas the C and F were the main two isoforms detected in ovary. Finally, high levels of the E hER{alpha} mRNA isoform were restricted to the liver (see upper strong band with E' probe and middle and strong band with F' probe), which confirmed the RT-PCR results previously published (31). It should be noted that no protected fragments corresponding to isoform D were obtained after the hybridization of probe D' with the different RNA samples followed by a S1 digestion even though it represented 8% of the clones selected by RACE performed on MCF7 total RNA and was readily detected by primer extension using a long primer (primer D). The S1 nuclease result does not exclude a major role for the isoform D since it may be more highly expressed in other tissues or at specific physiological stages.


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Table 1. Relative Abundance of hER{alpha} mRNA Isoforms in Different Human Cell Lines and Tissues

 
In light of the observations that estrogen down-regulated ER{alpha} expression in a variety of estrogen-target cells such as MCF7 (12, 24), the effect of this steroid hormone on the expression level of the different hER{alpha} mRNA isoforms in MCF7 was investigated. Cells were grown in medium, without phenol red and containing charcoal-dextran-stripped calf serum to eliminate estrogenic sources, for 5 days before a 24-h treatment with estradiol. An S1 nuclease protection assay was performed to examine the effect of the hormone treatment using the single-stranded DNA probes A'–F' specific for each hER{alpha} transcript as previously described. In this experiment, the ubiquitin S1 probe was coprecipitated with the RNA samples and hER{alpha} S1 probes to normalize the level of hER{alpha} transcripts. Densitometric analysis of the results are presented in Fig. 6Go. In this study, estrogen treatment resulted in a 40–60% decrease of the different hER{alpha} mRNA isoforms. These data indicate that the estrogen down-regulation of hER{alpha} expression in MCF7 is probably mediated by a mechanism that affects all hER{alpha} mRNA isoforms in a similar manner.



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Figure 6. Effect of Estrogen on the Expression Level of the Different hER{alpha} mRNA Isoforms in MCF7 Cells

MCF7 cells were grown in phenol red-free medium supplemened with 5% charcoal-treated calf serum. After 5 days, cells were cultivated in the presence or absence of 10 nM estradiol during 24 h. Total RNA was isolated using a guanidinium thiocyanate-phenol-cloroform extraction method. S1 nuclease mapping assays of the different hER{alpha} mRNA isoforms were then performed on 30 µg total RNA as described in the legend of Fig. 5Go, with the single-stranded probes A'–F' coprecipitated with an ubiquitin probe. Autoradiographs from the S1 protection assay were quantified by scanning densitometry, and the values were normalized using ubiquitin mRNA level. The results are presented as percentage of the total hER{alpha} mRNA expression detected in untreated MCF7 cells.

 
Previous studies reported that, in contrast to MCF7, ER{alpha} expression in liver is up-regulated by estrogen (13). It was hypothesized, therefore, that the level of hER{alpha} mRNA should be higher in adult premenopausal females than in males. Consequently, a sex-related variation of the expression level of the main hER{alpha} mRNA isoform detected in this tissue (E hER{alpha} mRNA) was investigated. Hence, S1 nuclease mapping experiments were performed on total adult male and female liver RNAs. There was a specific variation of E hER{alpha} mRNA level in the contribution of this isoform to the total hER{alpha} mRNA levels expressed in male and females as shown by the 2- to 3-fold increase in the relative level of E hER{alpha} messenger [E hER{alpha} mRNA/({Sigma} - E) hER{alpha} mRNAs] in the liver of adult females compared with the male sample (Fig. 7Go).



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Figure 7. Relative Expression Level of E hER{alpha} mRNA Isoform in the Liver from Both Sexes

Thirty micrograms of total RNA from the liver of one adult male and two adult females were hybridized to the labeled S1 probes F' as described in the legend of Fig. 5Go and treated with S1 nuclease, and the resistant hybrids were separated on a sequencing gel. The protected fragment from E' and F' hER{alpha} mRNAs, as well as from ({Sigma}-E/F) hER mRNAs, are indicated. Autoradiographs from the S1 protection assay were quantified by scanning densitometry, and then the relative expression level of E hER{alpha} mRNA isoform was calculated [E hER{alpha} mRNA/({Sigma}-E) hER{alpha} mRNAs].

 
Altogether, these results demonstrated that the utilization of multiple promoters and differential splicing is one of the mechanisms used to modulate the expression of the hER{alpha} gene in a tissue-specific and developmental manner.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} is known to be widely distributed in reproductive as well as nonreproductive tissues, thereby mediating estradiol action on various important physiological functions (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). It is obvious that the expression of the corresponding gene should be subject to a variety of controls to ensure that the correct amount of the ER protein is available in the correct cells at the correct time. A priori, this could be achieved either by 1) having many different target sites for different cell-specific factors on a single promoter or 2) having multiple promoter regions that have the possibility of responding to distinct combinations of factors present in the different tissues. This present study demonstrates that the hER{alpha} gene uses the second option, with tissue specificity and expression levels defined in part by the fact that this gene is a complex genomic unit exhibiting alternative splicing and multiple promoter usage.

Overall Organization of the hER{alpha} Gene
Using a RACE methodology, we have isolated and characterized several new hER{alpha} cDNA isoforms and demonstrated that these hER{alpha} transcripts are produced from a single hER{alpha} gene by the use of multiple promoters. The hER{alpha} mRNA isoforms are generated by splicing of five alternative upstream exons or leader sequences (1B–1F) to a common acceptor site that is situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1(A) (5). Preliminary analysis of the 5'-organization of the hER{alpha} gene by genomic walking techniques, S1 nuclease mapping, and primer extension studies showed that each of the hER{alpha} mRNA isoforms is transcribed using an isoform-specific promoter. Four of these promoters (A–D) were located within the 4-kb region upstream of the ER translational initiation codon. Southern blot hybridization and genomic PCR experiments suggested that the two others (E + F) were much further upstream.

The conservation of the acceptor sequences of the exon 1A splice site in other species, such as rodent, chicken, or Xenopus (data not shown), suggests that the expression of the ER{alpha} gene in these species also involves complex alternative splicing of upstream exons. The existence of similar phenomena for the rat (r) ER{alpha} gene has recently been demonstrated by the identification in RACE experiments of new rER{alpha} 5'-cDNA ends diverging from each other immediately upstream from the predicted alternative splice site (35, 36, 37). Likewise, in chicken, isoforms of ER mRNA that vary in their 5'-UTR sequences have also been recently isolated in our laboratory (38). Although highly probable, the multiplicity of leader sequences remains to be demonstrated for other species.

Features of the hER{alpha} mRNAs and the Corresponding Promoters
As demonstrated by the RACE and RT-PCR experiments, the hER{alpha} mRNA isoforms contain common sequences from exon 8 up to the acceptor splice site position located upstream from the AUG of the hER{alpha} coding region in exon 1A but diverged from each other immediately upstream from this position. The presence of a stop codon in exon 1A, in the part common to all hER{alpha} mRNAs, and upstream and in-frame with the translational initiation codon of hER{alpha} protein precludes the possibility that any of the hER{alpha} RNA isoforms described in this paper alter the N-terminal sequence of the ER{alpha} protein. Therefore, all hER{alpha} mRNA isoforms encode a common ER{alpha} protein. Sequence analysis showed that several of the 5'-UTRs of the hER{alpha} mRNA isoforms contain short ORFs (sORFs) (Fig. 2Go and Refs. 5, 26). The significance of these sORFs remains to be elucidated, but similarly placed sORFs in other messengers, such as GCN4 or the BCR/ABL oncogene mRNA, have been shown to be involved in the translational control of their expression (39, 40). It should also be noted that all of the hER{alpha} 5'-UTR sequences could be folded, using the algorithm of Zuker and Stiegler (41), into more or less stable secondary structures [{Delta}G values were ~-40 (E hER{alpha} mRNA) to -100 (A hER{alpha} mRNA) kcal/mol], which could also have an impact on the stability of the isoforms. Therefore, one possible function of these alternatively spliced 5'-UTR exons might be the regulation of the hER synthesis by controlling the turnover and/or the translation efficiency of the hER{alpha} mRNA isoforms. In keeping with this hypothesis is the fact that the 5'-UTR sequences of A and C hER{alpha} mRNA isoforms are highly homologous with the identified 5'-UTR sequences of the chicken and rodent ER{alpha} mRNA, respectively (26, 42). Although transcript B was not known to exist when the initial comparison were performed between chicken and hER genomic sequences (42), the results presented in the study showed the existence of a conserved region that corresponds to the leader sequences of isoform B. This region was shown recently to be also transcribed in chicken (38). These data suggest that a functional role for some of the 5'-UTRs has been preserved during evolution. Sequence alignment of the 5'-UTR of the D, E, and F hER{alpha} mRNA isoforms with the known ER{alpha} 5'-UTR sequences from other mammalian species showed no significant homology.

An evolutionary conservation of transcriptional regulations of the ER{alpha} gene is also suggested by the high degree of homology that has been shown between the human A and B promoter regions and the chicken ER{alpha} promoters (38, 42) and between the human C promoter region and the rodent ER{alpha} promoters (26). To date, no similar homology has been found for the promoter sequences of D, E, and F isoforms. Computer-assisted analysis of these new promoter sequences revealed an half-estrogen-responsive element in F hER{alpha} mRNA promoter as well as consensus and degenerate sequences for the binding of AP1 complex in the promoters of D and F hER{alpha} mRNA, respectively. These data are of particular interest given the fact that, in addition to its capability to bind to the estrogen response element, ER{alpha} can also act in a protein/protein related manner with the AP1 complex formed by c-Fos and c-Jun (43) and thereby potentially autoregulate its expression. Further studies are obviously necessary to characterize fully the different promoters of the hER{alpha} gene to elucidate the transcriptional events controlling the hormonal- and tissue-specific expression of the different hER{alpha} mRNA isoforms.

Tissue Specificity
The results on the distribution of the hER{alpha} mRNA isoforms showed a differential pattern of expression of the hER{alpha} gene in human tissues and cell types. As previously suggested, the predominant form expressed in the mammary gland and in the breast cancer cell lines is isoform A, accounting for approximately 50% of the total hER{alpha} mRNA. Interestingly, this transcript (together with isoform B in MCF7) was more abundant in the ER-positive breast tumor cell lines MCF7 and T47D than in the healthy tissue, whereas the expression of other hER{alpha} mRNA isoforms was not significantly altered (see Table 1Go). This observation suggests that the high ER{alpha} expression level in these breast tumor cell lines might be due to an overactivity of the corresponding promoters. Corroborating with this hypothesis is the fact that two binding sites for ERF-1, a member of the AP2 family of developmentally regulated transcription factors, have been located in the 5'-UTR of exon 1A of the hER{alpha} gene and are required for its efficient expression (44, 45). This transcription factor is highly expressed in ER-positive breast carcinomas in contrast to normal human mammary epithelial cells, where low levels of ERF-1 protein were detected (44). The proximity of these two ERF-1 binding sites to the A and B promoter regions might explain the comparative increase in the activity of these two promoters in most of the ER-positive breast cancer cell lines. Previous studies with the MCF7 breast cancer cell line have reported either no change or up to a 60% decrease in hER{alpha} mRNA level after an estradiol treatment, depending on the estrogen treatment history of the cells (12, 24). The present study shows that estradiol down-regulates the expression of each hER{alpha} mRNA isoform in a similar manner (see Fig. 6Go). This result is consistent with the fact that the main mechanism responsible for the hER{alpha} mRNA down-regulation in MCF7 cells was shown to be posttranscriptional (12) and may involve the 3'-UTR that is common to all hER{alpha} mRNA isoforms (46).

In the endometrium, the expression pattern of the hER{alpha} transcripts differed from that in breast tissue due to an increase in the proportion of the C hER{alpha} mRNA isoform to 40% of the total hER{alpha} mRNA, whereas the expression level of the other isoforms remains relatively unchanged between endometrium and breast tissues. The importance of ER{alpha} expression in the endometrium had previously been shown by the phenotypic changes in the uteri of a mutant mouse line with a insertional disruption of the ER{alpha} gene (47, 48). Likewise, our results show a significant expression of ER{alpha} transcripts in human ovaries with a predominance of C and F hER{alpha} mRNA isoforms. In rat, examination of ER{alpha} mRNA expression at cellular level, by in situ hybridization, showed that ER{alpha} mRNA was expressed at a low level throughout the ovary with no particular cellular localization, in contrast to ERß mRNA, which was expressed preferentially in granulosa cells of small, growing, and preovulatory follicules (49). Interestingly, high amounts of the E hER{alpha} mRNA isoform were restricted to the liver, which suggests that this isoform is important in some specific aspects of hepatic ER{alpha} regulation. In agreement with this hypothesis, it has been reported that hER{alpha} gene expression was differently regulated by estrogen in liver compared with endometrium and breast tissue or cell lines (12, 13). Estrogen increased hER{alpha} mRNA expression level in the liver, whereas hER{alpha} mRNA expression was down-regulated in the two other tissues. In addition, the hepatic ER{alpha} level was shown to be approximatively 5 times lower in immature than mature mammalian females (50). Our results confirm these data by the detection of higher levels of hER{alpha} mRNA in female compared with male liver. In addition, the S1 nuclease protection experiment shows clearly that the relative level of the predominant hER{alpha} mRNA isoform detected in the liver (E hER{alpha} mRNA) is higher in adult females than males. These data indicate that the tissue-specific expression of hER{alpha} transcript in liver is probably linked to a differential promoter or/and 5'-UTR usage and are in keeping with different promoter requirements for this important gene.

Several reports have suggested that estrogens also contribute to skeletal muscle growth (19, 51). Using immunoblotting and immunofluorescence microscopy approaches, recent work demonstrated that rodent skeletal myoblasts contain the ER{alpha} (19). Our S1 nuclease data show that the human male skeletal muscle ER{alpha} was encoded mainly by the C hER{alpha} mRNA isoform and more weakly by the A isoform.

Since estrogen deficiency has been recognized to be a cause of postmenopausal bone loss, several studies have demonstrated the presence of the ER{alpha} protein and its mRNA in skeletal tissue, especially in the osteoblasts and osteoclasts, the two bone cell types involved in the formation and resorption of the bone mass (15, 16). To determine which of the hER{alpha} mRNA isoforms were expressed in osteoblasts, RT-PCR and S1 nuclease mapping analysis were performed using total RNA from two osteoblast cell lines, HOS TE85 and SAOS. The E and F ER{alpha} mRNA isoforms were detected in both cell lines by RT-PCR, but none of the isoforms were present at sufficient levels to give a signal using the S1 nuclease method in these preliminary experiments. The low amounts of ER{alpha} transcripts detected in this study in the osteoblasts might be explained by the fact that only a small population of the cells that served as a source of RNA expressed ER{alpha} because of the cell cycle-dependent regulation of the ER{alpha} gene (16). The fact that only E and F hER{alpha} mRNA isoforms are expressed in the two osteoblast cell lines might be related to this correlation between ER expression and the cell cycle in osteoblastic cells, and these may have control elements required for such a pattern of expression.

Finally, apart from the D hER{alpha} mRNA isoform, all hER{alpha} transcripts were detected in brain and pituitary. Nevertheless, S1 nuclease protection analysis was unsuccessful in measuring hER{alpha} mRNA expression probably due to a low proportion of ER-positive cell types in these tissues and the fact that the RNA samples used in the experiments were prepared from males. In situ hybridization studies on female tissues using specific probes may be more appropriate to investigate the differential expression patterns of the ER mRNA isoforms in the pituitary and nervous system.

Importance and Possible Applications of Findings
As previously mentioned, the ER{alpha} is a key component in the signal transduction pathways controlled by the ovarian hormone, estrogen. These pathways direct a variety of physiological processes, such as establishment and maintenance of female sex differentiation patterns, reproductive cycle and pregnancy, liver, fat, and bone cell metabolism, cardiovascular and neuronal activity, and embryonic and fetal development (1, 2, 3). It is also well established that some of these pathways influence several pathological processes including breast, endometrium, and ovarian cancers, osteoporosis, arteriosclerosis, and Alzheimer’s diseases and that estrogen has both desirable and harmful effects on these human pathological processes (2, 3, 4). By providing evidence that the hER{alpha} gene is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner, this study should have implications in the basic, applied, and clinical research that involves ER biology. It shows that it is not always appropriate to focus on the A promoter and its messenger that were initially described (5) because in some tissues other hER{alpha} transcripts are predominant and would be unaffected by approaches that target A hER{alpha} mRNA isoform only. It also suggests approaches in which the transcriptional or translational efficiency of particular hER{alpha} transcripts might be altered: 1) by targeting transcription factors that bind specifically to the promoters or 2) by designing antisense oligonucleotides that are specific to each transcript, while allowing another hER{alpha} transcript to be functional; in this way, the benefical ER{alpha}-mediated effects are provided.

Finally, heterogeneity in the 5'-ends of mRNAs generated by alternative promoter usage and splicing seems to be a common feature among the members of the steroid/thyroid hormone/retinoic acid receptor family since several members of this family have also been reported to be transcribed from multiple promoters in a tissue-specific and developmental manner (52, 53, 54, 55). It is obvious that the increasing complexity of the organization and expression of the genes coding for the members of this family, which is emerging from this and others studies, is an appropriate means of achieving the differential and spatio-temporal expression of these important transcription factors, which may account to a large extent for the pleiotropic effects of their corresponding ligands in a wide range of physiological processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Lines and Tissues
The MCF7, T47D, ZR75–1, HEPG2, HOS TE85, SAOS, and HeLa cell lines were maintained in DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% FBS (GIBCO-BRL), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C in a 5% CO2 incubator. Five days before an estradiol treatment of MCF7 cells, the medium was replaced with phenol red-free DMEM containing 5% charcoal-treated calf serum. Endometrium and ovary tissues removed from patients undergoing hysterectomy or oophorectomy were kindly provided by Dr. R. Derham and Professor M. J. Mylotte (Department of Obstetrics and Gynecology, University College Hospital, Galway).

RNA Isolation
Total RNA from cell lines and tissues was extracted with TRIzol (GIBCO-BRL) as described by the manufacturer. Human mammary gland total RNA (a pool of RNA from six 16- to 35 yr-old Caucasian females), human brain total RNA (derived from a 60-yr-old Caucasian male), human liver total RNAs (derived from adult Caucasian male and females), and human skeletal muscle total RNA (derived from a 32-yr-old Caucasian male) were purchased from CLONTECH (Palo Alto, CA). Human pituitary RNA (derived from a Caucasian male) was kindly provided by Professor J. Duval (Université de Rennes, Rennes, France).

RACE
Alternative 5'-variants of hER cDNA were cloned by an inverse PCR method (30). RT of MCF7 total RNA (10 µg) and second-strand synthesis were performed using a commercial kit (GIBCO BRL) as recommended by the manufacturer, except that the hER{alpha} gene-specific primer IV (5'-CTGGCCGTGGGGCTGCAGGAAA) was used instead of the usual oligo (dT) primer (see Fig. 3Go). Subsequently, the cDNA was circularized in the presence of T4 DNA ligase and submitted to 35 rounds of PCR amplification using the sense primer IX (5'-CGCAGGTCTACGGTCAGACC) and the antisense primer VI (5'-TTGGATCTGATGCAGTAGGGC) (See Fig. 3Go). PCR included a denaturation step at 94 C for 1 min, an annealing step at 50 C for 1 min, and an elongation step at 72 C for 1 min, after an initial denaturation at 94 C for 5 min and was followed by 7 min elongation. One percent of the initial PCR product was reamplified under the same conditions with the nested primers X (5'-ACTCAACAGCGTGTCTCCGAG) and VII (5'-GGTCCGTGGCCGCGGGCAG) (See Fig. 3Go). The PCR products were subcloned in the TA cloning vector pCRTM2.1 (Invitrogen, San Diego, CA), and colonies were screened with the oligonucleotides A2 (5'-GCTGCGTCGCCTCTAACCTC) and C2 (5'-CAAGCCCATGGAACATTTCTG), which are specific for exons 1A and 1C, respectively (see Fig. 4AGo for the location of these two primers), and the oligonucleotide VIII (5'-AAGGCTCAGAAACCGGCGGG), which is 3' to the splice site and recognizes a common sequence of all hER cDNA 5'-ends (see Fig. 3Go). Colonies that did not hybridize to the A2 and C2 oligonucleotide probes but hybridized to VIII probe were sequenced by the dideoxy chain termination method.

Genomic Walking
The isolation and characterization of genomic regions further upstream of exon 1D, 1E, and 1F sequences generated by the RACE technique were performed using the Vectorette II starter pack from Genosys Biotechnologies Inc. (U.K.) as recommended by the manufacturer. Human genomic DNA was digested with different restriction enzymes (BamHI, BglII, EcoRI, HindIII, and TaqI) and then ligated to the appropriate vectorette units to form vectorette libraries. Five microliters of each vectorette library reaction were then used in two rounds of 30-cycle PCR amplification. The 3'-primers and nested primers used for the amplification of the genomic sequences further upstream of exon 1D, 1E, and 1F were D3 (5'-TGGCTCTCTCAGGTGAAGA) and D4 (5'-GAAGAAGGGTAAAGATTGAT), E3 (5'-TGCTGATATTTGGTACCGCAGTCCC) and E4 (5'-GAGATCTTTGTGCTTACTCCTT), and F3 (5'-TTGAAGAGAAGATTATCACTA) and F4 (5'-CTGTCTTCTTATGCTATAGAA), respectively (See Fig. 3Go). The common 5'-primer and nested primer were specific for the vectorette unit and provided by the manufacturer. Both rounds of amplification were performed using the Expand long template PCR system (Boehringer Mannheim, Indianapolis, IN). Ten microliters of each PCR reaction were electrophoresed on a 1% agarose gel and tranferred to nylon membranes (Hybond N+, Amersham, Arlington Heights, IL) with 20x saline sodium citrate (SSC) as transfer solution. The membranes were incubated in a prehybridization buffer containing 6x SSC, 5x Denhart’s solution, 0.05% sodium pyrophosphate, 100 µg/ml sperm DNA, and 0.5% SDS, at 37 C for 1 h. Then, the membranes were hybridized in 6x SSC, 1x Denhart’s solution, 0.05% sodium pyrophosphate, 100 µg/ml yeast tRNA with the oligonucleotide probes D5 (5'-TGGCTCCTCCGTTGAATGTG), E5 (5'-TCTTTGGTAGCTACAGAATATAATT), and F5 (5'-TAGAATGGGCAGGAGAAAGGAG), respectively (See Fig. 3Go), which had been end-labeled using T4 polynucleotide kinase and [{gamma}-32P] ATP (3000 Ci/mmol). The most stringent wash was carried out for 20 min at 55 C in 6x SSC, 0.05% sodium pyrophosphate. The specific PCR products were then subcloned in the TA cloning vector pCRTM2.1 (Invitrogen) and sequenced. The genomic portion separating exon 1D from the 3.2-kb identified genomic region upstream from exon 1A (27, 32) was amplified by two rounds of PCR amplification using human genomic DNA and the primers D1 (5'-CACATTCAACGGAGGAGCCA) and C4 (5'-CCTGGGACACTATGCAGTTACTGA) and the nested primer D2 (5'-ATCAATCTTTACCCTTCTTC) and C5 (5'-AATGTTAATGATGCCATCATGCAAAT) (see Fig. 3Go). To check the specificity, the PCR product was hybridized with the oligonucleotide probe C6 (5'-AATACTGACTATGGAGAGAG) as previously described, and then subcloned and sequenced.

Modified S1 Nuclease Mapping and Primer Extension
Biotinylated single-stranded DNA templates were used to prepare highly labeled single-stranded DNA probes or long primers by extension from a specific primer by the T7 DNA polymerase in the presence of [{alpha}-32P]dCTP (3000 Ci/mmol) (28, 29). These probes or long primers were then hybridized with the appropriate RNA sample and subjected either to an S1 nuclease digestion or to a reverse transcriptase extension, respectively. The origin of probe A template was a genomic PCR product obtained by amplification using the upstream 5'-biotinylated primer A0 (5'-AGCGACGACAAGTAAAGTAAAGT) with the downstream primer IV (see Fig. 1AGo). To prepare the templates used to make probes C, A', B', C', D', E', and F' (see Figs. 1AGo and 5AGo), as well as ubiquitin S1 probe, RT-PCR reactions were performed. The ER upstream primers for amplification were C0 (5'-ACTCCCCACTGCCATTCAT) (see Fig. 1AGo), A2, B2 (5'-ATCCAGCAGCGACGACAAGT), C2, D2, E1 (5'-AGCCTCAAATATCTCCAAAATCT), and F1 (5'-TTCTATAGCATAAGAAGACAG), respectively (See Figs. 4AGo and 5AGo). The ER common downstream primer was primer IV. Ubiquitin primers were Ub1 (5'-GTAAAAACCCTTACGGGGAAG) and Ub2 (5'-ACCACCACGAAGTCTCAACAC). The RT-PCR products were subcloned downstream of T7 in the TA cloning vector pCRTM2.1 (Invitrogen), and then PCR reactions were performed using a biotinylated T7 primer with either primer IV for ER probes or M13 reverse primer for ubiquitin S1 probe. The templates for primers {Sigma}, A, B, C, D, and E/F preparation were obtained by RT-PCR using the common downstream primer IV with the biotinylated upstream primers {Sigma}1 (5'-ATGACCATGACCGTCCACAC), A1 (5'-CTCGCGTGTCGGCGGGACAT), B1 (5'-CTGGCCGTGAAACTCAGCCT), C1 (5'-TCTCTCGGCCCTTGACTTC), D1 and E/F1 (5'-AAGGAGTAAGCACAAAGATCTC) (primer specific for E and F hER{alpha} mRNA isoforms and located in exon 1E), respectively. Finally, probe B, D, E, and F templates were prepared by PCR using the biotinylated T7 primer with primer V (5'-TCTGACCGTAGACCTGCG) and, for each reaction, two partially overlapping templates (a + b) to link promoter sequences with the corresponding 5'-cDNA end: 1) the TA cloning vector pCRTM2.1 containing the genomic region further upstream of exon 1B, 1D, 1E, or 1F. For D, E and F, these regions were generated by the genomic walking technique (see previous section) and for B by genomic PCR using the primers B0 (5'-GCACACCCCATTCTATCT) and B3 (5'-ACTTGTCGTCGCTGCTGGAT) (see Fig. 3Go); 2) the corresponding RT-PCR product obtained utilizing the upstream primer B1, D1, E1, or F1 with the common downstream primer IV.

All biotinylated PCR products were bound to streptavidin-coated magnetic beads (Dynal, Norway) as recommended by the manufacturer, and the nonbiotinylated DNA strands were removed in 0.1 M NaOH. A, B, C, D, E, and F S1 probes and A, B, C, D, and E/F primers were obtained by extending the VI primer annealed to the corresponding biotinylated single-stranded template, whereas probes A', B', C', D', and E' and primer {Sigma} were prepared using primer V. Ubiquitin S1 probes was prepared by extending Ub2 primer. After elution of the single-stranded DNA probes by alkaline treatment and magnetic separation, 105 cpm of the probe or primer were coprecipited with 30 µg of total RNA and then dissolved in 20 µl of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA, pH 8), denatured at 65 C for 10 min, and hybridized overnight at 55 C. The S1 digestions and the reverse transcriptase extension were carried out as previously described (56), and the samples were electrophoresed through a denaturing polyacrylamide/urea gels.

RT-PCR Analysis
cDNAs were synthesized using 1 µg of total RNA from different origins, an oligonucleotide primer (I) from the 3'-UTR (exon 8) of hER{alpha} mRNAs (5'-TTGGCTAAAGTGGTGCATGATGAGG) (see Fig. 4AGo), and 50 U of Expand reverse transcriptase (Boehringer Mannheim) under the conditions recommended by the supplier. Of the 20-µl reverse transcriptase reaction, 2.5 µl were then used in two rounds of 30-cycle PCR amplification. The 5'-primers and nested primers used for A, B, C, D, E, and F hER cDNA amplification were A1 and A2, B1 and B2, C1 and C2, D1 and D2, E1 and E2 (5'-AATTATATTCTGTAGCTACCAAAGAAG), and F1 and F2 (5'-GAGTGATAATCTTCTCTTCAA), respectively (see Fig. 4AGo). The 3'-primer II (5'-ATTATCTGAACCGTGTGGGAG) and the nested primer III (5'-CGTGAAGTACGACATGTCTAC) were from the common 3'-region of all hER cDNAs, immediately upstream of the primer used for reverse transcription. Both rounds of amplification were performed using the Expand long template PCR system (Boehringer Mannheim) as recommended by the manufacturer. Five microliters from each reaction were analyzed on an 1% agarose gel and transferred to nylon membranes (Hybond N+, Amersham). The membranes were hybridized as previously described (see Genomic Walking) with the oligonucleotide probe Ex. 2 (5'-CCCTGGCGTCGATTATCTGAA) (see Fig. 4AGo).


    ACKNOWLEDGMENTS
 
We thank G. Gannon, S. O’Brien, T. Dandekar, and M. Kulomaa for their contributions to this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Frank Gannon, European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstraße 1, D-69012, Heidelberg, Germany. E-mail: Gannon{at}EMBL-Heidelberg.de

This work was supported by a European Molecular Biology Organization long-term fellowship (to G.F.), the Irish-American Partnership (C.G.), the Irish Health Research Board (M.K.), and the Irish Cancer Research Advancement Board.

1 The nucleotide sequences reported in this paper have been submitted to European Bioinformatics Institute Data Bank with accession numbers AJ002559, AJ002560, AJ002561, and AJ002562. Back

Received for publication January 27, 1998. Revision received August 14, 1998. Accepted for publication September 8, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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