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The Control of Expression of Chicken and Human Estrogen Receptor Genesa

CAROLINE POPE,b GILLES FLOURIOT,b MARY ROSE KENEALY,b PADRAIG NESTOR,b AND FRANK GANNONc

bNational Diagnostic Centre, University College Galway, IrelandcEMBL, Meyerhofstrasse 1, D-69117, Heidleberg, Germany

Estrogens are ubiquitous in vertebrates, being found in fish, reptiles, birds, andmammals.1 Their main function is to control growth and development in reproduc-tive tissues. They also influence other tissue-specific processes such as homeostasisin the kidney, embryogenesis, bone, liver and fat metabolism, and cardiovascularfunction. Estrogens exert their potent physiological effects by binding to a nuclearreceptor, the estrogen receptor (ER). This protein belongs to the steroid/thyroid hor-mone/retinoic acid receptor superfamily whose members act as ligand-inducibletranscription factors.2 Family members are organized into structurally defined do-mains A-F which control functions such as ligand binding (E), DNA binding (C) andtranscriptional activation (A/B and E).3 As a reflection of the variety of processescontrolled by estrogens, the ER is found in many tissues. Its expression levels varyconsiderably among these tissues as well as within a single tissue. Clearly the ex-pression of such an important gene is subject to a variety of controls to ensure thatthe correct amount of protein is available in the correct cells at the correct time indevelopment. These interesting features of expression led our laboratory to furthercharacterize chicken (cER) and human (hER) ER genes in the hope of obtaining abetter understanding of these events.

GENOMIC ORGANIZATION OF THE cER AND hER GENES

The hER genomic organization was determined several years ago.4 This gene isover 140 kb in length and the coding region is split into 8 exons and 7 introns. Thisstructure was shown to be highly conserved in other members of the steroid receptorgene family. To determine the genomic organization of the cER gene, a genomic li-brary in λgt WES was screened using cDNA probes that spanned the entire openreading frame of the cER.5 Six clones were isolated and labeled A-F (FIG. 1). Se-quencing these clones using specific ER oligonucleotide primers showed that thecER coding region is also split into 8 exons with sizes 658, 190, 117, 139, 134, 184,and 5119 base pairs, respectively. Comparison of the intron/exon boundaries showsthat each corresponds to a canonical splice consensus sequence. Alignment of thechicken intronic sequence with that of the hER shows that the degree of conservationis not influenced by the homology within the adjacent exons and that the sequence

aThis work was funded by the Irish American Partnership and an EMBO long-term fellow-ship.

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FIGURE 1. Organization of the chicken estrogen gene (cER). The 8 exons of the cERgene are shown as solid boxes (genomic DNA). The corresponding position of the eightexons (1–8) with respect to the ER cDNA is shown above (cDNA) with numbers at theborders referring to the nucleotides. The positions of the translation initiation (ATG) andtermination (TAA) codons are indicated. Shown above the cDNA is the division of thecER protein into 6 regions A-F together with the DNA (region C)- and hormone(region E)-binding domains. The position of the six individual clones (A-F) with respectto the genomic DNA is shown at the bottom. The position of the exon/intron boundariesis on the left.

135POPE et al.: ESTROGEN RECEPTOR GENE REGULATION

homology decreases rapidly in all introns (data not shown). These results show thatthe cER genomic organization is very similiar to that of the hER, suggesting that thecommon 8 exons encoding the receptor protein are conserved through evolution.However, recently, the ER gene organization of two fish (rainbow trout and Oreo-chromis aureus) was determined.6,7 These genes possess an additional intron (V)which separates the hinge sequences from those of the hormone binding domain. Asthis is not the case for the chicken and human, it suggests that this intron was lostduring evolution.

For the chicken, primer extension showed that a major transcriptional start site ispresent 198 base pairs upstream from the ATG. It follows that the promoter regionis immediately upstream of the first translated exon. This is similiar to the situationin the hER gene but differs from rat, mouse, and fish ER genes for which an addi-tional upstream untranslated exon has been found.8,9

ALTERNATIVE SPLICING/PROMOTER USAGE IN cERAND hER GENES

Gradually data have emerged that many members are transcribed in a tissue-spe-cific manner from several promoters using alternative splicing within the 5′ translat-ed and/or untranslated regions.10,11 This may account to, a large extent, for thepleitropic effect of their ligands in the control of multiple physiological processes.In the hER we and others have shown the existence of 2 additional upstream alter-native exons (exon 1′and 1″) which are spliced to a common site upstream of thetranslation start site contained in the previously determined exon 112–14 (FIG. 2).One of these 2 exons (exon 1′) is highly homologous with the 5′ untranslated up-stream exon described in mouse and rat ER genes. This alternative splicing eventgives rise to several mRNA isoforms that differ in their 5′ untranslated region andso do not affect the open reading frame of the receptor protein. These results indicatethe presence of additional promoter regions, suggesting that the hER gene may besubjected to complex regulation by differential promoter usage.

In view of these results we were interested in determining if a similiar 5′ alterna-tive splicing event occurs in the cER gene. A single-stranded DNA probe mappingthe 5′ extremity of the cER gene was prepared to confirm by SI mapping the start

FIGURE 2. Schematic illustration of the 5′ genomic organization for the hER gene.

136 ANNALS NEW YORK ACADEMY OF SCIENCES

site position and to characterize the putative splice site. Three protected fragmentswere detected after hybridization of the probe with oviduct RNA followed by SI nu-clease digestion, with their 5′ extremities located 198, 191, and 44 base pairs up-stream from the start of the chicken ER open reading frame. No protected fragmentswere seen with tRNA used as a negative control. The 5′ positions of the two longestfragments correspond to the transcription start site already characterized.5,15,16 Theshortest fragment is due to partial protection of the probe up to 44 base pairs up-stream of the ATG. The relative abundance of these three fragments is approximate-ly 35%, 35%, and 30%, respectively. An identical pattern but with weaker intensitywas observed after SI nuclease mapping of liver ER mRNA with the same probe.

These results show that a fraction of the cER mRNA has 5′ extremities differentfrom those of the proximal 5′ end and indicates that splicing from upstream regionsis a significant event in the expression of the cER gene (FIG. 3). These findingsprompted us to investigate further using a RACE technique (rapid amplification ofcDNA ends) to amplify these new 5′ ends.17 In this way four new 5′ untranslatedupstream exons were found, each of which splices at a common position upstreamof the translation start site defined by the SI nuclease experiment, thus giving newcER mRNA isoforms. The new chicken upstream sequences are not homologous tothose found previously in the hER gene. Analysis of 3 kb of known 5′ cER upstreamsequence reveals that these new sequences are not contained within it. We have start-ed to screen a chicken cosmid library to determine the exact genomic location ofthese new exons.

FIGURE 3. SI nuclease mapping reveals that a portion of the cER mRNAs present dif-ferent 5′ extremities. As depicted in the diagram, a probe mapping a part of exon 1 andthe promoter regions of the cER gene was hybridized with total RNA from oviduct, liver,and tRNA and subjected to SI nuclease digestion. Products were sized by running on apolyacrylamide gel next to M13 control sequence. Arrows indicate the location of twostart sites and the splice site.

137POPE et al.: ESTROGEN RECEPTOR GENE REGULATION

CONCLUSIONS

This study demonstrates that the cER gene is similiar to other members of the nu-clear receptor family and also that it is a complex genomic unit. It exhibits alterna-tive splicing of at least 4 upstream untranslated 5′ exons which are under the controlof different promoters. Further investigation will give vital information on the con-tribution of these promoters to the regulation of the cER gene. This is an importantstep for our understanding of the different spatial and temporal expressions of thecER gene in estrogen target tissues and will enrich similiar ongoing studies with thehER gene.

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