Two Functionally Different Protein Isoforms Are Produced from the ...

Two Functionally Different Protein Isoforms Are Produced from the Chicken Estrogen Receptor-a Gene

Caroline Griffin, Gilles Flouriot, Vera Sonntag-Buck, and Frank Gannon European Molecular Biology Laboratory (C.G., G.F., V.S-B., F.G.) D-69117, Heidelberg, Germany National Diagnostic Centre (C.G.) University College Galway, Ireland

The existence of two forms of the chicken estrogen receptor-a protein (ER-a) in chicken tissues is demonstrated: the previously reported receptor (cER-a form I), which has a size of 66 kDa, and a new form (cER-a form II), which lacks the N-terminal 41 amino acids present in form I and thus gives rise to a protein of 61 kDa. Whereas the 66-kDa protein is the translation product of several cER-a mRNAs (A1–D), the cER-a protein isoform II is encoded by a new cER-a mRNA (A2), which is transcribed in vivo from a specific promoter that is located in the region of the previously assigned translation start site of the cER-a gene. SI nuclease mapping analysis reveals that cER-a mRNA A2 is liver enriched. The resulting cER-a forms I and II differ in their ability to modulate estrogen target gene expression in a promoter- and cell type-specific manner. Whereas cER-a form I activates or represses in a strictly E2-dependent manner, the truncated form is characterized by a partial transactivating or repressing activity in the absence of its ligand. Comparison of the N-terminal coding regions of different vertebrate ER-a reveal a conservation of the translation start methionine of the protein ER-a form II in other oviparous species but not in mammals. The expression of two classes of ER-a transcripts encoding the two ER-a receptor forms in the liver of Xenopus laevis and rainbow trout is demonstrated. Therefore, the existence of two functionally different protein isoforms produced from the ER-a gene is probably a common and specific feature in oviparous species. (Molecular Endocrinology 13: 1571–1587, 1999)

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INTRODUCTION Estrogen is an important regulator of reproductive functions and cell division (1, 2). In oviparous species, the liver is one of the main estrogen target tissues. In the maturing female chicken, the onset of vitellogenesis involves hepatic synthesis of a group of egg yolk proteins including very-low-density apolipoprotein II (apoVLDL II) and the vitellogenins. When estradiol is provided to the chicken, the complete set of yolk genes can be induced in the liver of both male and females (3). The oviduct is a second estrogen target tissue in oviparous species. Administration of estradiol to immature chicks triggers the cytodifferentiation of tubular gland cells located in the magnum position of the oviduct, which synthesize the major egg white proteins, ovalbumin, conalbumin, ovomucoid, and lysozyme, and modulates the rate at which oviduct proteins are synthesized (Ref. 4 and references therein). The physiological changes induced by estrogens often result from modifications in the expression patterns of specific genes and are mediated through specific nuclear proteins, the estrogen receptors (ERs). These receptors belong to a large family of ligandactivated transcription factors whose members, the steroid, thyroid hormone, and retinoic acid receptors, regulate gene expression by interacting either in a protein/DNA manner with cognate DNA sequences called responsive elements (Ref. 5 and references therein) or in a protein/protein manner with other transcriptional factors (6). Structure-function analysis of these receptors reveals that they are modular proteins composed of a DNA-binding domain (region C) and a hormone-binding domain (region E) (7, 8). In addition, two other regions have been shown to mediate their capability to transactivate target genes. A transcriptional activation function 1 (AF-1) has been located in the A/B domain and functions as an independent transcription activating domain in yeast and mammalian cells. A hormone-inducible transcription activating domain, AF-2, is present in the hormone-binding domain. 1571

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Both AF-1 and AF-2 are required for optimal stimulation of transcription, but the relative contributions of the two varies in a cell- and promoter-specific manner (9–11). Diversity in the family of receptors that respond to the same ligand is generated in many ways. In some instances, receptors are encoded by separate genes present at discrete genetic loci. These include the retinoic acid receptors RARa, -b, and -g (12–15) and RXRa, -b, and -g (16, 17), the thyroid hormone receptors, TRa and TRb (18), and the two estrogen receptors, ERa and ERb, found in mammals (19–21). For the same gene, differential promoter usage and alternative splicing may also generate receptor isoforms, differing either at the N terminus (22–24) or at the C terminus (25). A relevant illustration of this is the progesterone receptor (PR), which exists as two isoforms, PR-A and PR-B. These forms are produced from distinct promoters and differ at their N-terminal end. The human PR-A lacks the first 164 amino acids of PR-B while sharing the remainder of the coding region of the gene. An indication of the possible biological significance of protein isoforms lies in the demonstration that the two PR isoforms have different efficiencies in inducing progesterone-responsive element-mediated responses depending on the promoter and the cellular contexts (26–28). Both human and chicken ERa genes have been shown recently to generate several mRNA variants (A–F, hERa mRNAs; and A–D, cERa mRNAs) by alternative splicing of upstream exons to a common site upstream of the translation start site (29, 30). All of these transcripts differ in their 59-untranslated regions (59-UTR) but code for the same ERa protein. In this paper we report the existence of a second cERa protein referred to as cERa form II (61 kDa in size). This form lacks the first 41 amino acids present at the N terminus of the previously characterized full-length cERa form I (66 kDa in size). The I and II forms of the cERa receptor can be produced in vitro and are also detected in vivo in oviduct and liver tissues. We show that each cERa protein isoform originates from distinct mRNA classes (mRNAs A1–D and A2) that are transcribed in vivo from different promoters. The tissuespecific expression of both cER-a mRNA species A1–D and A2 was analyzed and shown to vary in the different chicken tissues tested. Electrophoretic mobility shift assays demonstrated that cER-a form II is able to bind to an estrogen-responsive element (ERE) in vitro, but in contrast to cER-a form I, the N- terminal truncated form can, to a limited extent, modulate estrogen-responsive promoter activity in an E2-independent manner in vivo. Finally, comparison of the amino acid sequence of the N-terminal coding regions of different vertebrate ER-a proteins indicates that the two ER-a forms exist in other oviparous species but not in mammals. We show that in the frog (Xenopus laevis) and rainbow trout (Oncorhynchus mykiss), distinct ERa mRNA transcripts may encode both ERa protein forms I and II. The possibility that the two ERa

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forms could play different roles in the control of gene expression by estrogens in oviparous species is discussed.

RESULTS Existence of Novel Liver-Enriched cERa Transcripts Whose 5*-Ends Are Located Downstream of the Previously Assigned Translation Start Site The expression level of cERa mRNAs was compared in two main estrogen-responsive tissues in chicken, oviduct, and liver, by a SI nuclease mapping analysis using probes that covered either the 59-extremity of the gene including the transcription start site of the A1 mRNA isoform (11) and exon 1B–1D acceptor splice site in exon 1A (1154) (probe A), or a part of the region coding for the hormone-binding domain (probe B) (Fig. 1A). Quantitative analysis of the results showed that the intensity of the cERa-specific signal obtained with total RNA from oviduct was comparable using both probes (data not shown). However, a clear difference was found in the signal level from the two probes when liver total RNA was used. The signal detected by probe B mapping a part of the hormone-binding domain was greater in intensity than that found using probe A mapping the 59-extremity of the gene, although both probes were similar in their size and specific activity (data not shown). These data indicated that new cER-a mRNA variant(s), resulting from either transcriptional initiation(s) or an alternative splicing event(s) that occur between the two regions mapped by probes A and B, contribute in part to the total cERa mRNA content in liver. To locate either of these new transcription start site(s) or alternative splice site(s) within the coding part of the cERa gene previously delimited, another SInuclease mapping experiment was performed using probe C, which was complementary to cER-a mRNA A1 sequences from 1158 to 1892, as schematically depicted in Fig. 1A. A protected fragment of 734 nucleotides, corresponding in size to the fully protected probe, was obtained from total RNA of both laying hen oviduct and liver tissues, while no signal was visible with yeast total RNA used as a negative control (Fig. 1B). This fragment was expected from many previous experiments and results from a protection of cERa mRNA isoforms A1–D. In addition to this band and a smear of minor specific bands that are probably caused by hybridization of cERa mRNAs with partially degraded SI probes (see the pattern of the free probe), two other major specific products of 607 and 588 nucleotides, respectively, mainly present in liver RNA sample, were detected. The 59-extremities of these protected fragments are located in a region downstream from the initiator methionine position, at 1285 and 1304 in exon 1A. The fact that the same two sites were subsequently mapped by a primer extension

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Fig. 1. Existence of Novel Liver Enriched cER-a Transcripts Whose 59-Ends Are Located Downstream of the Previously Assigned Translation Start Site A, Schematic representation of the cER-a transcripts. The original cER-a mRNA is encoded by eight exons labeled 1–8. The position of the two initiator methionines (ATG1, codon 1; and ATG2, codon 42) and the termination codon (TAA) are indicated. The division of the cER-a protein into six regions, A–F, together with the DNA-binding (region C) and hormone-binding (region E) domains are shown directly above the cDNA. The location of the splice acceptor site at 1154 in exon 1A is also marked. Three alternative upstream 59-noncoding exons (B, C, and D) splice to this position giving rise to cER-a mRNA isoforms 1B–1D. Probe A (from 2169 to 1318), B (from 11298 to 11678), and C (from 1158 to 1892) used for SI nuclease mapping are indicated. For primer extension analysis, a primer complementary to positions 1360 to 1572 of the cER-a cDNA was used. B, Preliminary evidence for a new cER-a mRNA (A2) using SI nuclease mapping analysis. One hundred micrograms of total RNA from laying hen oviduct and liver were hybridized to a labeled probe C and treated with SI nuclease as described in Materials and Methods. Yeast RNA was used as a negative control. The SI nuclease-resistant hybrids were separated on a 4% polyacrylamide gel adjacent to DNA mol wt markers and free probe. The position of the two cER-a mRNA A2 transcription start sites are indicated on the right side of the figure. Also indicated are the relative positions of ATG1 and ATG2. C, Mapping of the cER-a transcription initiation sites by primer extension. Fifty micrograms of total RNA from laying hen oviduct and liver were hybridized to the long primer and treated with reverse transcriptase, and the extension products were separated on a sequencing gel (as described in Materials and Methods). The transcription start sites of A1 and A2 cER-a mRNAs are indicated. Also indicated are the relative positions of ATG1 and ATG2. D, The pattern of distribution and the relative levels of the two classes of cER-a transcripts, S A1–D cER-a mRNAs and A2 cER-a mRNAs, were determined by SI nuclease mapping assays using total RNA from various sources as indicated at the top of each lane. M and F indicates male and female samples, respectively. Yeast RNA was used as negative control. Protected fragments are marked with arrows. The relative abundance of the two classes of cER-a mRNA transcripts are shown below each lane of Fig. 1D. The values were calculated from the densitometric scanning of the protected fragments obtained after SI nuclease analysis and expressed as the percentage of the total cER-a mRNA expressed in the oviduct. 1/2 indicates that a weak expression of cER-a mRNA was observed in a minority of the analyzed RNA samples.

analysis (Fig. 1C), again most notably in the liver RNA sample, excludes the possibility that they resulted from a splicing event. Therefore, the new mRNA species (called cERa mRNA A2 to distinguish them from the previously characterized cERa mRNA A1) are transcriptionally initiated within exon 1A of the cERa gene at positions 1271, 1285, and 1304.

Examination of the cDNA sequence downstream of the newly determined transcription start sites showed the presence of an ATG codon at position 1347/9 (methionine 142), which is in-frame with the remainder of the cERa open reading frame (Fig. 1A). Analysis of the sequence surrounding this ATG (59-GCGACATGT39) revealed a favorable Kozak sequence for transla-

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tion initiation (31). Therefore, this ATG could function as a translation initiation codon for cER-a mRNA A2 and thus would give rise to a truncated 547 amino acid cERa protein with a predicted size of 61 kDa. The level and pattern of distribution of cER-a mRNA A2 were analyzed in a panel of chicken tissues using SI probe C. The results show that these transcripts are mainly expressed in liver and ovary (Fig. 1D). It should be noted however that a weak expression of cERa mRNA A2 was observed in a minority of the analyzed oviduct RNA samples (data not shown). The fully protected fragment that is specific to the remainder of the cERa mRNA isoforms (S A1–D cERa mRNAs) was visible as expected in oviduct, liver, ovary, and lung. Using probe C, it was not possible to find a significant expression level of S A1–D cERa mRNAs in kidney and testis. Densitometry of the signals obtained in this experiment allowed a quantitative comparison of the expression levels of A2 cERa mRNA with those found for S A1–D cERa mRNAs. Summarized in a table below Fig. 1D, these data are expressed as a percentage of the total cERa mRNA expression detected in oviduct using the S1 probe C. This comparative analysis revealed a tissue specificity in the level of expression of the A2 cERa mRNA isoform. The liver is the main site of expression for this isoform, although weak expression was detected in ovary and occasionally in oviduct. A Functional Promoter Maps the Region of the Previously Assigned Translation Start Site of the cERa Gene To investigate whether sequences located upstream of the transcription start site of cERa mRNA A2 exhibited promoter activity, three fragments from 2503 to 1183, 2503 to 1381, and 132 to 1381 were generated by PCR using as a template a genomic l clone containing exon 1A and 3 kb of sequence upstream of the 59-end of the cERa cDNA (32). These fragments were subcloned upstream of the luciferase reporter gene of the pGL2 basic vector, thus generating the reporter vectors, pGL2503/1183, pGL2503/1381, and pGL132/1381, as schematically illustrated in Fig. 2A. A reporter plasmid pGL(GH4) containing 802 bp of nonspecific DNA was also constructed for use as a size control for the transfection experiments. These vectors and the parental vector pGL2 basic were transiently transfected into chicken embryo fibroblast (CEF) cells. Compared with the size control and the empty parental vector, each cERa gene-specific fragment was able to initiate transcription as determined by an increase in luciferase activity (Fig. 2B). The highest activity was observed with pGL132/1381, demonstrating that the region located in the vicinity of the transcription start sites for cERa mRNA A2 contains a functional promoter, called promoter A2 hereafter. When this region was extended to include sequence from promoter A1 (pGL2503/1381), a 1.5-fold decrease in promoter activity was observed. This effect

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may be attributed to the putative silencer elements located between 2437 and 2384 in this second promoter (32). Finally, promoter A2 was able, in part, to counterbalance the activity of this silencer as a further 1.5-fold reduction in activity was observed when promoter A2 region was removed from construction pGL2503/1381 giving rise to pGL2503/1183 (Fig. 2B). Two cERa Protein Isoforms I and II Are Produced in Vivo As previously mentioned, the examination of the cDNA sequence downstream of the transcription start sites of cERa mRNA A2 showed the presence of an ATG codon at position 1347/9 (methionine 142), which is in frame with the remainder of the cERa open reading frame (Fig. 1A). To test whether this in-frame ATG could function as a translation initiation codon for a truncated cERa protein with a predicted size of 61 kDa, cER-a cDNAs were inserted in pSG5 to generate the expression vectors pSG cER-a I and pSG cER-a II. The expression vector pSG cERa II, which contained sequences from 1308 to 12038, was expected to generate a cERa protein in which translation was initiated at methionine 42, while vector pSG cERa I, composed of the original cERa cDNA (from 1158 to 12038), should produce cERa proteins initiating at methionine 1, and methionine 42 if there is leakiness in the translation initiation (Fig. 3A). The hERa cDNA expression vector (HEO) was used as a positive control (33). Analysis of the expression of constructs pSG cER-a I and HEO in a rabbit reticulocyte lysate system in the presence of radioactively labeled methionine showed 66-kDa ERa protein, as expected (Fig. 3B). Interestingly, a second smaller cERa protein was observed from the expression vector pSG cERa I whose size correlated to a truncated cERa receptor protein starting at methionine 42, a similar leakiness of translation is noted for HEO with an alternative ATG used to generate a minor protein band. The second cERa form was the only form produced by the expression vector pSG cERa II, thus confirming the initiating translational functionality of the in-frame ATG codon at position 1347/9 (methionine 142) (Fig. 3B). This new protein was called cERa form II relative to the full-length receptor cER-a form I. To check the existence of the cER protein forms I and II in vivo, a Western blot analysis was performed using chicken oviduct and liver nuclear extracts. Protein extracts were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with the monoclonal antibody H 222 directed against the ligand-binding domain of the hERa protein (34). The result confirmed the expression in both tissues of two cERa proteins of the expected molecular mass, 66 and 61 kDa (Fig. 3C). To compare the levels of the two cERa forms, the protein signals from the immunoblot were quantified by densitometry. An 8- to 10-fold excess of the cERa form II over form I was

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Fig. 2. A Functional Promoter Maps the Region of the Previously Assigned Translation Start Site of the cER-a Gene A, Diagrammatic illustration of the luciferase reporter constructs used in the experiment: pGL2503/1183, pGL2503/1381, and pGL132/1381. The transcription start sites of cER-a mRNA A1 and A2 are represented by broken arrows. Exon 1A is indicated by an open box. Also indicated is the position of ATG1 and ATG2 and finally a black box represents the position of the putative silencer element (2437 to 2384) (32). B, Constructs pGL2503/1183, pGL2503/1381, and pGL132/1381 were transiently transfected into CEF cells as described in Materials and Methods. Two promoterless luciferase reporters, the empty vector pGL2 basic and pGL2 basic containing an 802-bp fragment of nonspecific DNA [pGL(GH4)], were included as negative controls. The luciferase activity of each transfected construct was assessed and corrected for transfection efficiency using an internal reference plasmid EF-1a-CAT. Values correspond to the average of three separate transfection experiments 6 SDs and are expressed as a percentage of the pGL2 basic luciferase activity.

observed in the liver, whereas cERa protein I was the major form expressed in the oviduct (5 times higher). Finally, the presence of another protein, recognized by this H 222 antibody, should be noted. This protein of apparent molecular mass of 45 kDa corresponds most likely to initiation of translation at an internal methionine (Meth. 170) within the coding region of the cERa cDNA.

cERa Form II Protein Binds Specifically to an ERE As the DNA-binding domain of cERa protein form II is identical in sequence to the original cERa, it was expected to bind with similar efficiency to the same type of estrogen response element (ERE), namely an inverted repeat spaced by three nucleotides (GGT-

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CAnnnTGACC). Electrophoretic mobility shift assays with both chicken ERa receptors were conducted to confirm this assumption. Figure 4 shows that cERa protein isoforms produced by the rabbit reticulocyte lysate system are indeed able to form retarded complexes in the presence of the radiolabeled consensus ERE from the apoVLDL II promoter (35). The specificity of these complexes was confirmed by competition experiments using equal, double, or a 10-fold excess

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of unlabeled consensus apoVLDLII-ERE, since a reduction and ultimate elimination of the ER-ERE complex signal were observed. In addition, a 10-fold excess of a mutated ERE had no effect. The complexes had different mobilities depending on the extract used. The slower migrating complex (A) was obtained from the extract producing cER-a form I (66 kDa), whereas the faster one (C) is produced from cERa form II (61 kDa) extract. Finally, an intermediary mobility complex

Fig. 3. Two Protein Isoforms I and II of cER-a Gene Are Produced in Vitro and in Vivo A, Schematic representation of the cDNAs inserted within the expression vectors pSG cER-a I, pSG cER-a II, and HEO and encoding the cER-a form I, cER-a form II, and hER-a proteins. The position of the two initiator methionines (ATG1, codon 1; and ATG2, codon 42) and the termination codon (TAA) are indicated. In the expression vector pSG cER-a II, the cER-a sequences between nucleotides 1158 and 1308 were not included. Therefore, the sequences preceding ATG2 are noncoding. B, pSG cER-a I, pSG cER-a II, and HEO plasmids were in vitro transcribed and translated by rabbit reticulocyte lysate in the presence of [35S] methionine. Translation products were resolved on a 10% SDS-polyacrylamide gel and sized relative to the migration of prestained molecular size markers. C, Nuclear protein extracts from chicken oviduct and liver were separated in parallel on the same gel and then subjected to immunoblotting with the H 222 antibody. Immunoreactive bands 66 and 61 kDa in size were visualized by ECL.

Fig. 4. cER-a Form II Protein Binds Specifically to an ERE cER-a receptor form I, form II, and a combination of both receptors produced from rabbit reticulocyte lysate (RRL) were incubated with 1 ng of labeled apoVLDLII-ERE. Specificity was determined in the absence (2) or presence (1) of equal, double, or 10-fold amounts of unlabeled apoVLDLII-ERE competitor (flattened triangle) or a 10-fold amount of unlabeled mutant ERE (m) as a nonspecific competitor. Control reactions containing unprogrammed RRL in the presence or absence of radiolabeled ERE were used as negative controls. The positions of the three specific cER-a DNA complexes (A–C) are indicated by arrows. A corresponds to form I homodimer-ERE complexes; B, form I and form II heterodimer-ERE complexes; C, form II homodimers-ERE complexes. An asterisk indicates a faster migrating nonspecific complex.

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was generated when both receptor forms were synthesized simultaneously from the rabbit reticulocyte lysate. Therefore, complex A and C presumably correspond to homodimers of the cERa form I and II, respectively, while complex B is probably due to the formation of cERa form I and II heterodimers. Also worth emphasizing is the fact that when both receptor forms are coproduced, no cERa form I homodimer formation is visible. This is probably accounted for by a cERa form I/II ratio of approximately 1:2. Differential Transactivation Capability by cERa Form I and II To ascertain whether distinct functional properties can be attributed to the two receptor forms, their ability to transactivate chimeric estrogen- responsive genes containing complex endogenous promoters such as those for the vitellogenin II (VTG II) (36) and the apoVLDL II (35) genes, which are expressed specifically in the liver of laying hens, was studied. The reporter genes used were luciferase (LUC) or chloramphenicol acetyltransferase (CAT), yielding the reporter constructs VTGII-LUC and apoVLDLII-CAT. These constructions were cotransfected into the CEF and chicken hepatocellular carcinoma (LMH) cell lines grown in the presence or absence of E2, with the expression vectors that selectively express either form I or II of the cER-a. As expected from previous work (37), the ability of E2 to induce the VTG II promoter was found to be cell specific since a hormone- and receptor-dependent induction was observed only in LMH cells (Fig. 5). No difference in the transactivation efficiency (10- to 20fold increase) of VTG II promoter was detected between cER-a form I and II proteins in these cells (Fig. 5). However, results showed that whereas no transactivation of the VTG II promoter by either of the two liganded cER-a forms was observed in CEF cells, the unliganded cER-a form II was able to repress the basal activity of VTG II promoter (3-fold decrease). Results of a similar nature were obtained using the second chimeric estrogen-responsive apoVLDL II gene. This construct was induced in the presence of E2 to a similar level (100- to 200-fold increase) in both cell types in the presence of the two cER-a forms I and II (Fig. 5). In contrast, whereas the transactivation of the apoVLDLII-CAT reporter gene by cER-a form I is hormone dependent in both cell lines, the N-terminal truncated cER-a form II is able to partially transactivate it in a significant manner in the absence of its ligand (10- to 20-fold increase). It should be noted that, in LMH cells, the induction of apoVLDLII-CAT activity measured in the presence of estradiol, but in the absence of cotransfected cER-a receptor, may be explained by the presence of endogenous cER-a in this liver-derived cell line. We conclude from these data that the cER-a form II, unlike cER-a form I, possesses a hormoneindependent transactivation activity that functions in a promoter-dependent manner.

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As estrogen has been shown previously to be involved in the regulation of expression of the cER-a gene (3, 38), it was also interesting to study the effect of the two cER-a protein isoforms on the promoters that control their production. Therefore, the reporter constructs pGL(GH4), pGL2503/1183, pGL2503/ 1381, and pGL 132/1381 described earlier (see Fig. 2A) were transfected with the expression vector pSG5, pSG cER-a I, pSG cER-a II, or pSG RARa (negative control) in CEF cells. The data showed that each of the three promoter fragments was down-regulated in a ER-a-dependent manner as shown in Fig. 6. Moreover, as previously found for the apoVLDLII-CAT reporter gene, the truncated form of the cER-a was able to function in absence of estradiol whereas the fulllength receptor required the ligand to down-regulate the transcriptional activity of the cER-a promoter fragments. As all three promoter constructs reacted in a similar manner to the presence of receptor, we deduced that the cis-acting element mediating this effect was probably in a region common to all three promoter fragments and is therefore between 132 and 1183 in the cER-a gene. The N-Terminal Truncated ER-a Form II Receptor Is Conserved in Oviparous Species but Not in Mammals A comparison of the N-terminal primary amino acid sequence of human (h) (19), mouse (m) (39), rat (40), chicken (c) (41, 38), Xenopus laevis (x) (42), and rainbow trout (rt) (43, 44) ER-a proteins demonstrates that the first initiator methionine codon, called ATG1, is conserved in all species analyzed with the exception of rainbow trout (Fig. 7). Translation from ATG1 results in the production of the classical 66-kDa ER-a protein. In contrast, the original rtER-a cDNA, cloned from a rainbow trout liver cDNA library, encodes an N-terminal truncated ER-a protein that is translated from an initiator methionine similarly positioned to the second translation start site (ATG2) used to produce the liverenriched cER-a form II protein. ATG2 is also conserved in X. laevis but is absent in human, mouse, and rat ER-a receptors. In mammals, this downstream methionine codon was replaced by a valine that is unable to initiate translation (Fig. 7). These data suggested the likely existence of previously unidentified ER-a protein forms in rainbow trout and X. laevis, equivalent to the 66- and 61-kDa cER-a forms, respectively. If indeed this was the case, then the production of these two ER-a protein forms from a single ER-a gene might be a conserved feature of oviparous species. To check this hypothesis, an SI nuclease mapping analysis was performed to investigate the existence of distinct classes of ER-a transcripts in X. laevis and rainbow trout that might encode two protein forms of ER-a, similar to the classical full-length cER-a form I and the N-terminal-truncated cER-a form II. The SI probes for the chicken and X. laevis ER-a genes used in this experiment were designed to cover exon 1 and

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Fig. 5. Transcriptional Activation of the Estrogen-Responsive VTG II and apoVLDL II Gene Promoters by cER-a Forms I and II CEF and LMH cells were transiently transfected with 5 mg of the reporter plasmid VTGII-LUC or apoVLDLII-CAT, together with 0.5 mg of the expression vector pSG5, pSG cER-a I, or pSG cER-a II. Cells were treated with or without estradiol (1028 M) for 24 h before being assayed for luciferase and CAT activities. Results are expressed as a percentage of the reporter activities (VTGII-LUC or apoVLDLII-CAT activities) measured in the presence of the expression vector pSG cER-a II, in the absence of E2. Luciferase and CAT activities were normalized using the internal reference controls, EF-1a-CAT and pCMV-tk-LUC, respectively. Values correspond to the average of at least two separate transfection experiments 6 SDs.

a part of its 59-flanking region as depicted in Fig. 8A (42, 45). Regarding the rainbow trout ER-a gene, it was previously shown that the assigned translation start site was located in the second exon, which was separated from the untranslated first exon by an 846-bp intron 1 (46, 47). However, the analysis of the 59genomic flanking sequence of exon 2 revealed an in-frame initiator methionine, 45 amino acids upstream of the previously characterized initiator codon (this upstream translation start site in intron 1 will be called ATG1 and the initiator codon in exon 2 will be renamed ATG2 hereafter) (see Fig. 8A). This new organization of potential translation start points in the rtER-a gene

was therefore comparable to that described herein for the chicken. To ascertain whether this 59-intronic flanking sequence of exon 2 was transcribed, a specific rainbow trout SI probe was designed to protect this region and a part of exon 2 (see Fig. 8A). Finally, to allow for easier comparison of the SI nuclease mapping results, the three ER-a probes were designed to contain the same distance (286 nucleotides) between their 39-end and ATG2. The results of the SI nuclease mapping analysis of chicken liver RNA shown in Fig. 8B confirmed those described earlier. Four protected fragments representing the transcription start site of the cER-a mRNA A1

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Fig. 6. Autoregulation of the cER-a Promoters pA1 and pA2 by cER-a Forms I and II Constructs pGL2503/1183 (pA1 promoter), pGL132/1381 (pA2 promoter), and pGL2503/1381 (pA1 and pA2 promoters) were transiently cotransfected into CEF cells with the expression vector pSG5, pSG cER-a I, or pSG cER-a II. The expression vector pSG RARa containing the mouse retinoic acid receptor a cDNA was included as a negative control. Also included as negative controls were the two promoterless luciferase reporters, the empty vector pGL2 basic and pGL2 basic containing an 802-bp fragment of nonspecific DNA [pGL(GH4)]. Cells were treated with or without estradiol (1028 M) for 24 h before being assayed for luciferase activities. The reporter gene activities were normalized according to the activity of the cotransfected EF-1a-CAT control. Values correspond to the average of two separate transfection experiments, which showed no significant difference and are expressed as a percentage of the pGL2 basic luciferase activity.

transcript, the splice site at position 1154, and the two transcription start sites for cER-a mRNA A2 were detected. In the case of X. laevis, two protected fragments of approximately 520 and 350 nucleotides in size were also specifically observed after SI nuclease analysis of liver RNA from females in vitellogenesis (Fig. 8B). The 59-extremity of the longest was positioned upstream of the translation initiation codon (ATG1) position, whereas the 59-end of the shorter fragment was located in a region between ATG1 and the putative initiator methionine codon ATG2. These two positions were also mapped by a primer extension experiment (performed as described earlier for the chicken ER-a gene) (data not shown), thereby demonstrating that they correspond to transcription start sites of distinct xER-a mRNAs, which potentially encode different xER-a protein forms of 66 and 61 kDa. It should be noted that no fragments corresponding to a protection which extends to the previously described transcription start site of the xER-a gene were detected (45). Neither was a partial protection of the Xenopus SI probe as far as a position equivalent to the

alternative splice site found in exon 1 of the cER-a gene observed, even though sequence analysis reveals a good candidate acceptor site (ctgttttcag/GTG) at a similar position in exon 1 of xER-a gene. Finally, as for the two previous oviparous species investigated, the results of the SI nuclease mapping analysis of rainbow trout liver RNA pointed to the existence of transcripts that potentially encoded the different ER-a protein forms. As expected, the previously described rtER-a mRNA (43, 44), as well as alternative splicing variants (47), was partially protected by the SI probe up to the 59-extremities of exon 2 due to the noncomplementarity of their 59-end (the untranslated exon 1) to the probe (see asterisks in Fig. 8B). This transcript has the potential to encode the previously described rtER-a protein (N-terminal truncated form). The SI nuclease mapping experiment showed, in addition, the existence of new rtER-a transcripts which initiate in a region of intron 1 at approximately 40 and 100 nucleotides upstream of the predicted in-frame initiator methionine (ATG1), as confirmed by a primer extension experiment (data not shown). The translational prod-

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Fig. 7. Comparison of the N-terminal Primary Amino Acid Sequence of Vertebrate a-ERs Reveals a Second Translation Start Codon Which Is Highly Conserved in Oviparous Species The ClustalW (1.7) program (69) was used for the multiple amino acid sequence alignment of human (hER-a), mouse (mER-a), rat (rER-a), chicken (cER-a), X. laevis (xER), and rainbow trout (rtER) ERs. Input sequences were retrieved from the Swiss Protein database. Gaps (2) were introduced into the sequence to obtain maximum alignment of identical amino acids. Asterisks indicate conserved residues in all species. Arrows mark the location of initiator methionines (M) (ATG1 and ATG2). The highly conserved C region, which encodes the DNA binding domain of the nuclear receptor family, is boxed.

uct of this new class of transcripts should be a longer form of rtER-a protein equivalent to the 66-kDa cER-a receptor. The conclusion of these SI nuclease mapping analysis is that the liver tissue of the oviparous species tested, chicken, X. laevis, and rainbow trout, express distinct classes of ER-a mRNAs able to generate either the normal ER-a protein or an N-terminal truncated form.

DISCUSSION ER-a is known to be widely distributed in reproductive as well as nonreproductive tissues, thereby mediating estradiol action on various important physiological functions that range from female sexual development and reproduction, to liver, fat, and bone cell metabolism (1–3). It is obvious that the expression of the ER-a

gene should be subject to a variety of controls to ensure that the correct amount of ER-a protein is available in the correct cells at the correct time. Therefore, the elucidation of the molecular mechanisms controlling the tissue-specific pattern of ER-a gene expression should provide a starting point to understanding the pleiotropic effects of its ligand in a wide range of biological processes. Recent studies on the structure and organization of the human and chicken ER-a genes showed that these genes are complex genomic units exhibiting alternative splicing and promoter usage in a tissue-specific manner (29, 30). In both species, all identified ER-a mRNA isoforms diverged in their 59-UTR sequences upstream from the translational initiation codon and therefore encoded a common ER-a protein of 66 kDa in size. In this present study, we demonstrated that the chicken ER-a gene is able to produce a second cER-a protein of 61 kDa in size, called cER-a form II. As

Two Protein Isoforms I and II of the Chicken ER-a Gene

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Fig. 8. Identification of Distinct Classes of ER-a mRNA, Which Potentially Encode Either the Full-Length or the N-Terminal Truncated ER-a Protein Forms in the Liver of Different Oviparous Species A, Schematic representation of the approximate location of the chicken (probe D), X. laevis, and rainbow trout probes used for the SI nuclease mapping experiment as well as the genomic regions mapped by these probes. Preparation of the probes has been described in Materials and Methods. The position of the two initiator methionines (ATG1 and ATG2) and the location of the transcription start site previously described for the three species are indicated. It should be noted that for the rainbow trout ER-a gene, the assigned translation start site is located in the second exon, which is separated from the untranslated first exon by an 846-bp intron. B, One hundred micrograms of total RNA prepared from liver tissue in vitellogenesis of female chicken, X. laevis, and rainbow trout, together with 100 mg of yeast RNA (as a negative control) were hybridized to the corresponding uniformly labeled SI probe and treated with SI nuclease, and the resistant hybrids were separated on a 6% polyacrylamide gel next to an aliquot of free probe and end-labeled mol wt marker for sizing. Diagrammatically indicated on one side of the gel is the protected fragments corresponding to the transcription start sites (11), splice sites (*), and the translation start sites ATG1 and ATG2.

shown by its in vitro production using the rabbit reticulocyte lysate system, this second form results from initiation of translation at methionine 42 and thus lacks the 41 amino acids present at the N-terminal domain

of the 66-kDa cER-a form I. Two mechanisms might be involved in the production of these two proteins. Both isoforms may be translated from the same transcripts as a consequence of the leaky ribosome-scan-

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ning mechanism (31), or their production could arise from distinct mRNA species, each of which encodes a different protein. Whereas in vitro and in vivo translation of the previously identified cER-a mRNAs (A1–D) generated both forms of cER-a protein (see Fig. 3, B and C, oviduct sample), which indicates that the leaky ribosome-scanning mechanism may occur, SI nuclease mapping and primer extension analysis of liver total RNA allowed the existence of another class of cER-a mRNA transcripts (A2) to be shown, in agreement with the second mechanism. This new class of cER-a transcripts is transcribed from positions 1285 and 1304 in exon 1A, between the translation initiation methionine 1 and methionine 42. Therefore, these transcripts are unable to encode the cER-a protein form I. Their translational product is the truncated receptor form II. Sequence analysis of the 59- UTR region of A2 cER-a mRNA revealed that this region is devoid of short open reading frames (sORFs) in contrast to the 59-UTRs of A1–D cER-a transcripts, which contain at least one sORF (30). The significance of these sORFs remains to be elucidated, but similarly placed sORFs in other messages such as the GCN4 and the BCR/ABL oncogene mRNA have been shown to be involved in the posttranscriptional control of their expression (48, 49). Therefore, further experiments should be informative on the existence of a differential turnover and/or translational regulation of the two classes of cER-a mRNAs. The 59-flanking genomic region of the new A2 cER-a transcripts was shown to be able to promote the transcription of a luciferase reporter gene in transient transfection experiments performed in the CEF cell line, thus providing evidence that a previously unknown promoter (pA2) was located in exon 1A regions coding for the N-terminal part of cER-a protein form I. These data demonstrated clearly that cER-a form II can be specifically generated from transcripts distinct from those encoding form I and that a unique promoter is responsible for the production of cER-a form II only. In contrast to the previously characterized pA1 promoter, which contains a well positioned typical TATA sequence, with potential CAAT box sequences close to the start site of transcription (32), the promoter of A2 cER-a transcripts is devoid of any obvious TATA or CAAT-box sequences. Computer- assisted analysis of promoter A2 sequences failed to identify any consensus EREs (50). This is in contrast to pA2 transfection results in CEF cells, which demonstrated clearly its down-regulation by E2 in an ER-a-dependent manner and therefore suggests that protein/protein interaction is involved. Comparable data were previously obtained for the pA1 promoter (30). As pA1 and pA2 promoters are located close to each other, both promoters may be similarly affected by the cis-acting element(s) involved in the autoregulation as suggested by transfection studies herein. These results have to be integrated with the reports that cER-a gene expression is differently regulated by estrogen in liver and oviduct tissues. Estrogen increases cER-a expression

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level in the liver (3), whereas cER-a mRNA expression is down-regulated in the oviduct (38). Interestingly, promoter pA1 is the main promoter used to transcribe the cER-a gene in oviduct tissue while promoter pA2 activity is predominant in the liver and weak or absent in oviduct. Therefore, the mechanism by which a single ligand, estradiol, mediates such opposing effects in distinct tissues probably involves complex interactions between cis-acting element(s) and tissue-specific factor(s). Further studies, such as the analysis of the DNAse I-hypersensitive sites, followed by a more detailed promoter characterization using the in vivo footprinting technique would be informative in identifying sequences involved in this cell-specific expression of the cER-a mRNAs. Analysis of the pattern of expression of the two classes of cER-a mRNAs (A1–D and A2) revealed that their relative levels vary in the different chicken tissues. As previously reported (30), the highest amount of A1–D cER-a mRNA class was detected in the oviduct, and lower amounts were present in liver and ovary, two other chicken female reproductive tissues tested. In contrast, expression of the second cER-a mRNA class was mainly observed in liver, was weakly detected in ovary, and, in most of the samples studied, was absent in oviduct. The consequence at the protein level of this differential distribution pattern of the two cER-a mRNA classes should be that tissues expressing the first class of cER-a mRNAs will only contain both cER-a protein forms as the result of the leaky scanning mechanism (31) while tissues that coexpress A2 cER-a mRNA transcripts will produce the truncated receptor as the main cER-a protein form. Therefore, the presence of A2 cER-a mRNA class in a tissue should allow the ratio between the two cER-a proteins to be changed in favor of form II. Immunoblot analysis of chicken oviduct and liver nuclear extracts confirmed this hypothesis since both isoforms were detected in oviduct and liver tissues with the truncated cER-a form II predominantly produced in liver tissue where it accounted for approximately 80% of the total immunoreactive ER-a. Two distinct classes of ER-a mRNAs potentially encoding the normal and N-terminal truncated form of the ER-a protein were also detected by SI nuclease mapping analysis in the liver of other oviparous species such as X. laevis and rainbow trout. In contrast to a previous investigation (51), which proposed that the production of the two forms of xER-a was the result of the leaky ribosome-scanning mechanism (31), the present study provides evidence that a new class of xER-a mRNAs initiate downstream of the previously determined translation start site (ATG1) in a region coding for the N-terminal part of xER-a protein form I. Likewise, the initially characterized rainbow trout ER-a mRNA encodes a protein equivalent to the N-terminal truncated receptor form II. The present study demonstrated the existence of a second class of rtER-a mRNAs in rainbow trout, whose translation product is a receptor possessing 45 additional amino acids at its

Two Protein Isoforms I and II of the Chicken ER-a Gene

N-terminal domain. Western blot analysis, previously performed using an antibody raised against the rainbow trout ER-a protein, confirmed the presence of two receptor forms in rainbow trout liver (52). However, the origin of these two proteins was not determined in that study (52). Comparison of the N-terminal primary amino acid sequence of vertebrate ER-a proteins reveals the conservation of the second translation start codon (ATG2) in oviparous species (chicken, X. laevis, and rainbow trout) but not in mammals (human, mouse, and rat). These data indicate that the existence of two different protein isoforms produced from the ER-a gene is probably a common and specific feature in oviparous species and suggests that the two ER-a forms could play different roles in the control of gene expression by estrogens in these species. The two ER-a protein forms I and II identified in this report show similarity with the A and B forms of the chicken and human PR (26, 53, 54). The PR form A is an N-terminally truncated variant of PR form B that may arise by translation initiation at an internal methionine codon of the PR mRNA (55) or by translation of a specific PR mRNA (54). Several groups have reported that PR form A and B have distinct functions and influence gene transcription differently depending on the promoter and cell context. For instance, Tora et al. (26) found that chicken PR form B is entirely inactive on the chicken ovalbumin promoter, in a setting in which form A is a strong transactivator, while the MMTV promoter was activated more efficiently by form B than form A. In addition, human PR form A is a stronger transactivator than form B on the tyrosine aminotransferase promoter (27). Likewise, the present study showed that the two chicken ER-a forms differ in their ability to modulate transcription activity of estrogen target genes in a promoter- and cell type-specific manner. Whereas cER-a form I activates or represses in a strictly E2-dependent manner, the truncated form is characterized by a partial activity in the absence of its ligand. Supporting this result is the previous observation that the rainbow trout ER-a, which is equivalent to the N-terminally truncated cER-a form II, exhibits a basal transcriptional activity in yeast, while no activity was measured for the human ER-a (full-length ER-a form) (56). This functional difference between the human and rainbow trout ER-a, or between the two chicken ER-a forms, is probably due to the presence or absence of the N-terminal A domain in the receptor protein. Further structure function studies will be necessary to elucidate the exact role played by this A domain in mediating target gene activity and especially its impact on the two transcription activation functions (AF-1 and AF-2) of the ER. The evolutionarily conserved production of a functionally different ER-a isoform in the liver of oviparous species (but not in mammals) suggests a key role for this form in the production of very-low-density lipoproteins and/or egg yolk proteins such as vitellogenin. Vitellogenesis is an important estrogen-initiated process unique to the liver of oviparous vertebrates (57).

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Previous studies aimed at providing insights into the molecular events involved in vitellogenesis and its regulation have employed the full-length ER-a cDNA whose translational product is the receptor form I (66 kDa) (3, 58). The data presented in this paper demonstrate that this is not a true representation of the in vivo hepatic environment where the main ER-a mRNA isoform expressed encodes an ER-a protein devoid of its N-terminal 41 amino acids. Gene transfer technology may be helpful in providing an answer to the interesting question of whether there are physiological consequences resulting from blocking the production of the truncated ER-a form II by mutation of methionine at position 42 (ATG2). In conclusion, the production of two ER-a forms from a single ER-a gene in oviparous species provides two different transacting regulatory proteins which, in addition to their respective spatial distributions, differ in their ability to modulate the activity of estrogenresponsive genes. This new ER-a complexity may account, to a large extent, for the pleiotropic effects of the corresponding ligand, estradiol, in a wide range of physiological processes occurring in oviparous vertebrates.

MATERIALS AND METHODS RNA Isolation Six-month-old laying hens (Rhode Island Red, European Molecular Biology Laboratory, Heidelberg, Germany) and adult cock were killed. The oviduct, liver, ovary, lung, and kidney from hens, and the liver and testis from cock were removed immediately and frozen in liquid N2. Liver was also removed from female X. laevis and rainbow trout in vitellogenesis. Total RNA was extracted from all tissues with TRIzol (Life Technologies, Inc., Gaithersburg, MD) as described by the manufacturer. Modified SI Nuclease Mapping and Primer Extension Modified SI nuclease protection and primer extension procedures were followed as described by Flouriot et al. (59, 60). These two methods involved the use of biotinylated singlestranded DNA templates to prepare highly labeled singlestranded DNA probes or long primers by extension from specific primers with the T7 DNA polymerase in the presence of [a-32P]dCTP (3000 Ci/mmol). These probes or long primers are then hybridized with the appropriate RNA sample and subjected either to an S1 nuclease digestion or to a reverse transcriptase extension, respectively. To prepare the template used to make probe C (from 1158 to 1892) (see Fig. 1A), an RT-PCR reaction was performed. The 59- and 39-primers used for the amplification were S1 (59-ACTGCCAGCTGCCGATCTTG-39) and S2 (59-ATAGTACACTGGTTAGTGGCAG-39), respectively. The RT-PCR product was subcloned downstream of T7 and upstream of M 13 reverse primer in the TA cloning vector pCR 2.1 (Invitrogen, San Diego, CA). A PCR was then performed using a biotinylated T7 primer with M 13 reverse primer. The origin of the chicken (probe D), X. laevis, and rainbow trout probe templates was genomic PCR products obtained by amplification of the regions from 2232 to 1633 (32) (see Fig. 8A), from 2243 to 1632 (42, 45) (see probe D in Fig. 8A),

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and from 2424 in intron 1 to 1508 in exon 2 (46, 47) (see Fig. 8A). The DNA fragments were then subcloned in the pCR 2.1 vector (Invitrogen) downstream of T7 and upstream of M 13 reverse primer. A PCR reaction was then performed using a biotinylated T7 primer with M 13 reverse primer. The template for the long cER-a primer was obtained by RT-PCR using the 59-biotinylated S3 (59-GCAACAAGACAGGAGTTTTTAACTA-39) and the 39-primer S4 (59-CTGTAGAAGGCTGGAGGAGCAGCT-39). All biotinylated PCR products were bound to streptavidincoated magnetic beads (DynAl) as recommended by the manufacturer (Dynabeads, Dynal, Hamburg, Germany), and the nonbiotinylated DNA strands were removed in 0.1 M NaOH. The S1 probes (C and D, Xenopus and rainbow trout probes) and the long primer were obtained by extending their respective primer [S2, S5 (59-CCCTCATCCCAAAGCTGCCCTGT-39), X1 (59-TTACTGCGAAAGTGCCCTGCTCAC-39), RT1 (59-CCAGGTAGTATGACTGGCTGG-39) and S6 (59-ATGGATGAAGGGTGAGAGCTG-39), respectively] annealed to the corresponding biotinylated single-stranded template. After elution of the single-stranded DNA probes by alkaline treatment and magnetic separation, 105 cpm of the probe or primer were coprecipited with 100 or 50 mg, respectively, of total RNA and then dissolved in 20–30 ml of hybridization buffer (80% formamide, 40 mM piperazine-N,N9-bis(2-ethanesulfonic acid), pH 6.4, 400 mM NaCl, 1 mM EDTA, pH 8), denatured at 70 C for 10 min, and hybridized overnight at 55 C. The S1 digestions and the reverse transcriptase extension were carried out as previously described by Ausubel et al. (61), and the samples were electrophoresed through denaturing polyacrylamide/urea gels. Expression Vector Preparation pSG cER-a I was prepared as described by Griffin et al. (30). By the same approach, the expression vector pSG cER-a II was made by directionally cloning the cER-a coding region from 1308 to 12038 into the parental expression vector pSG5 (62). HEO [pSG5 expression vector containing the complete hER-a cDNA (33)] and pSG RARa [pSG5 expression vector containing the mouse retinoic acid receptor a cDNA] were gifts from P. Chambon. In Vitro Transcription and Translation In vitro transcription and translation were accomplished with the TNT-coupled Reticulocyte Lysate system from Promega Corp. (Madison, WI) following the manufacturers directions. pSG5 recombinant expression vectors pSG cER-a I, pSG cER-a II, and HEO were used as templates for transcription with T7 RNA polymerase followed by translation to generate cER-a I, cER-a II, and hER-a proteins. Translation efficiency was checked by incorporating [35S] methionine. Two microliters of the radioactive translation product were run on a 10% SDS-PAGE gel as outlined by Laemmli (63). The gel was dried and autoradiographed. Cold methionine was used in the in vitro transcription and translation of proteins for electromobility shift assays and Western blot analysis. Nuclear Extract Preparation Nuclear extracts were prepared from freshly killed egg-laying hen oviduct and liver tissues using the procedure of Sierra et al. (64). Protein concentration was determined using the Bradford protein assay solution from Bio-Rad Laboratories, Inc. (Richmond, CA). Western Blot Analysis Nuclear protein from egg-laying hen oviduct and liver tissues and 7.5 ml of in vitro transcription and translation mix were

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subjected to SDS-PAGE. Proteins were denatured at 95 C for 15 min and resolved on a 10% SDS polyacrylamide gel next to prestained broad-range protein standards from Bio-Rad Laboratories, Inc. and electrotransferred to Immobilon nitrocellulose membrane (Millipore Corp., Bedford, MA). The membrane was blocked in TS (10 mM Tris, pH 7.4, 0.5 M NaCl) containing 10% (wt/vol) nonfat dry milk powder. The membrane was incubated with primary antibody (2 mg/ml)-anti hER-a monoclonal antibody H 222 kindly provided by Dr. G. L. Greene (34) in TS containing 3% nonfat dry milk powder for 1 h at room temperature. Incubation with secondary peroxidase-coupled goat antirat antibody was performed under the same conditions. ER-a proteins were visualized by chemiluminesence using the ECL system from Amersham Pharmacia Biotech (Arlington Heights, IL) according to the manufacturers instructions. Signals were quantified by densitometry. Electrophoretic Mobility Shift Assay Chicken ER-a I and ER-a II proteins were prepared by in vitro transcription and translation as described above. One microliter of in vitro translated product was preincubated at 0 C for 20 min in binding buffer (10% glycerol, 10 mM Tris, pH 7.5, 1 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 0.1 M KCl) in the presence of 80 mM Mg/Spermidine, 2 mg of poly dI:dC, 2.5 mg sonicated salmon sperm DNA, and 10 mM Na2HPO4. The samples were then incubated with 1 ng of radioactive oligonucleotide probe [50,000 dpm] end labeled with [g-32P] ATP (3000 Ci/mM) using T4 polynucleotide kinase. Competition was performed by premixing different concentrations of unlabeled competitor oligonucleotide with radioactively labeled oligonucleotide before addition to the binding reaction. Protein-DNA complexes were separated from free probe by nondenaturing electrophoresis on a long 5% polyacrylamide gel in 0.25 3 TBE. The gel was prerun at 4 C for 60 min followed by 5 h running at 600 V. After electrophoresis the gel was fixed for 30 min in a 10% methanol/acetic acid solution and dried before autoradiography. Sequence of the consensus ERE 30-bp oligonucleotide was derived from the 59-flanking region of the chicken apoVLDL II gene (2186 to 2156) (65). The nucleotide sequence was 59-ctgtgctcaGGTCAGACTGACCttccatta-39 with the wild-type consensus ERE sequence shown in uppercase letters. The sequence of a mutant version of this oligonucleotide (m) was 59-ctgtgctcaGGACACTGTGTACttccatta-39. The mismatches are underlined. Both oligonucleotides were used as double-stranded DNA for the electrophoretic mobility shift assay. Promoter Construct Preparation To create the luciferase reporter plasmids pGL2503/1183, pGL2503/1381, and pGL132/1381, PCR was used to amplify, from a genomic l clone containing 3 kb of sequence upstream of the 59-end of the cER-a cDNA (33), regions from 2503 to 1183, 2503 to 1381 and 132 to 1381, respectively. For each construction, the two synthetic primers used for the amplification [pAI (59- TGCGCTGGTACCTCTTTTACATTCTTCAATTTCTG-39) and pAII (59-AGTGCGAAGCTTCAACAGCAAGATCGGCAGCTGG-39), pAI and pBII (59CGTGCGAAGCTTTAAAAACTCCTGTCTTGTTGCTT-39) or pCI (59-GTTCATGGTACCCCAGTGCTCACCCTGCATTTGT39) and pBII, respectively] were designed to introduce 59 KpnI and 39 HindIII restriction sites (underlined within the primer sequence) at the ends of the PCR products. The amplified fragments were directionally cloned into the polylinker of the pGL2 basic plasmid (Promega Corp.) upstream of a luciferase reporter gene. pGL(GH4) reporter plasmid containing 802 bp of nonspecific DNA was also constructed for use as a size control for transfection experiments. The following CAT-containing reporter plasmids were kind gifts: The apoVLDL II gene promoter fragment from 2900 to

Two Protein Isoforms I and II of the Chicken ER-a Gene

11455 (apoVLDLII-CAT) from M. Evans (35) and the chicken vitellogenin II promoter fragment from 21133 to 111 from J. Burch (36). This was subsequently subcloned into pGL2 basic vector to give the construct VTGII-LUC. Cell Culture and Transient Transfection Assays CEF cells (a gift from T. Graf, Heidelberg, Germany) were maintained in DMEM supplemented with 5% FCS, 1% chicken serum, 10 mM HEPES, pH 7.4, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in a 5% CO2 incubator. LMH cells (ATCC, Manassas, VA) were grown in Weymouth’s MB/251 medium with 10% FCS, L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. CEF cells were transiently transfected using the DNA/calcium phosphate coprecipitation method (66). LMH cells were transiently transfected as described by Binder et al. (37). In all transfection studies, 6-cm dishes containing 5 3 105 cells were transfected with a total of 10 mg of DNA per dish [5 mg reporter plasmid, 1 mg of expression vector, 0.1 mg of internal control (EF-1a-CAT or pCMV-tk-LUC) (67), and carrier DNA to 10 mg (pBluescript)]. Medium was changed 6 h before transfection. After 16 h incubation with the DNA/calcium phosphate precipitate, the medium was aspirated and cells washed twice with PBS, and fresh serum-stripped phenol red free medium was added. Transfected cells were cultured for 24 h in the absence or presence of 1028 M 17b-estradiol before harvesting for luciferase and CAT assays. Luciferase assays were performed as outlined by Brasier and Ron (68) on 20% of the lysate. The CAT activity was assayed for with the ELISA kit from Roche Molecular Biochemicals (Mannheim, Germany) using 20% of the lysate. The reporter gene activity values were normalized for transfection efficiency according to the activity of the cotransfected reference control (EF-1a-CAT or pCMV-tk-LUC).

Acknowledgments Received January 14, 1999. Revision received May 6, 1999. Accepted May 24, 1999. Address requests for reprints to: Frank Gannon, European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012, Heidelberg, Germany. E-mail: Gannon @ EMBL-Heidelberg.de. This work was supported by the Irish American Partnership fellowship to Caroline Griffin, the Irish Cancer Society, and by an European Molecular Biology Laboratory long-term fellowship to Gilles Flouriot.

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