Human Estrogen Receptor Gene Research Articles

The 3'untranslated region of the human estrogen receptor gene post-transcriptionally reduces mRNA levels.

The 3'untranslated region of the human estrogen receptor gene post-transcriptionally reduces mRNA levels.

Association of Estrogen Receptor Dinucleotide Repeat Polymorphism with Osteoporosis

We investigated the association between dinucleotide (thymine-adenine) repeat polymorphism lying upstream of human estrogen receptor (ER) gene and bone mineral density (BMD) as well as biochemical markers for bone metabolism in 144 healthy postmenopausal Japanese women. The genotype was classified into "A" through "R" according to the number of the repeats, from 10 to 27. BMD was expressed in Z score (a deviation from the weight-adjusted average BMD of each age using the standard deviation as a unit). The people having genotype C (12 repeats of thymine-adenine) allele (n=15) had significantly lower Z score of spine BMD (mean ± SD; −1.11 ± 1.3 vs. −0.06 ± 1.2; p<0.01) and of total body BMD (−0.58 ± 1.0 vs. 0.31 ± 0.9; p<0.01) than those without this genotype (n=129). They also had significantly higher levels of serum intact osteocalcin, urinary pyridinoline, and urinary deoxypyridinoline. These results suggest that genetic variation at the ER locus may be associated with some determinants for BMD and bone metabolism in postmenopausal women.

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene.

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (+/- 1,341) 17beta-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (+/- 2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased approximately 25-fold in hFOB/ER9 cells following 10(-9)M E2 treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10(-9)M E2 resulted in a 2.5-4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function.

Alteration of the expression of human estrogen receptor gene by distamycin

The effects of distamycin on the expression of the estrogen receptor gene were determined in the MCF7 human breast cancer cell line. Estrogen receptor (ER) RNA transcripts were analyzed by Northern blotting and RT-PCR using specific oligonucleotides for the 5′ upstream region and for ER cDNA. After ex vivo distamycin treatment of the cells the expression of the canonical ER mRNA isoform of 6.3 kb is strongly inhibited, without appreciable alteration of the accumulation of 5′ upstream ER mRNA isoforms. These results suggest that distamycin alters the transcriptional activity of the ER gene causing a change in the ratio between the canonical transcript and other isoforms containing 5′ upstream regions.

Mutational analysis of the estrogen-receptor gene in endometrial carcinoma

To determine whether mutation in the DNA of the estrogen-receptor gene occurs in endometrial cancer. Polymerase chain reaction amplification and single-stranded conformation polymorphism analysis of the entire coding region (exons 1-8) of the human estrogen-receptor gene, as well as an untranslated region (exon I*) in the gene, were performed on genomic DNA extracted from 56 snap-frozen endometrial cancers. All cancers demonstrating mobility shifts on single-stranded conformation polymorphism suggestive of DNA sequence alteration were subjected to definitive DNA sequencing of the relevant portion of the estrogen-receptor gene. In addition to detecting a frequent, previously described polymorphism in exon 1, single-stranded conformation polymorphism analysis of the 56 endometrial cancers identified seven cancers with mobility shifts. Three cancers shifted in exon 3, one cancer each shifted in exons 4 and 7, and two shifted in exon 8. Deoxyribonucleic acid sequencing revealed sequence alterations in all seven cases demonstrating mobility shifts. In six of these seven cases, these alterations were consistent with infrequent silent polymorphisms; in the seventh cancer, the sequence alteration proved to be a somatic missense mutation at codon 537 in the region of the estrogen-receptor gene encoding the hormone-binding domain of the receptor protein. The infrequent DNA mutation in the estrogen-receptor gene is unlikely to account for the variation in estrogen-receptor expression observed in endometrial cancer.

Differential effects of distamycin analogues on amplification of human gene sequences by polymerase-chain reaction.

In this report we analyse the effects of distamycin and five distamycin analogues on amplification by polymerase-chain reaction (PCR) of two gene sequences displaying a different A+T/G+C content. The first was a 5' region of the human oestrogen receptor (ER) gene, containing a (TA)26 stretch; the second was a CG-rich sequence of the human Ha-ras oncogene. The results obtained unequivocally demonstrate that the addition of one pyrrole ring significantly improves the ability of distamycin derivatives to interfere with PCR-mediated amplification of the human ER genomic region carrying a (TA)26 stretch. The distamycin analogues analysed differ in the number of pyrrole rings and in the presence of an N-formyl, an N-formimidoyl or a retroamide group at position X1. Among compounds carrying the same number of pyrrole rings, those carrying an N-formyl or an N-formimidoyl group retain a similar inhibitory activity. The retroamide analogues, on the contrary, are much less efficient in inhibiting PCR-mediated amplification of the 5'ER region. With respect to sequence selectivity both distamycin and distamycin analogues exhibit a sequence preference, since they do not inhibit PCR amplification of Ha-ras CG-rich gene regions, with the exception of a distamycin analogue carrying four pyrrole rings.

Generation and characterization of a human osteosarcoma cell line stably transfected with the human estrogen receptor gene.

Although 17 beta-estradiol (E2) replacement therapy has been shown to be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional endogenous estrogen receptor (ER) in some osteoblastic cells has been demonstrated, and the suggestion that the abundance of ER may be rate-limiting in the action of E2 on these cells has been made. To study the mechanism of ER in regard to E2-mediated effects, we stably transfected a human osteosarcoma cell line, SaOS-2, with an expression vector, pMV-7-ER, containing the human ER gene. We characterized six of the stably transfected clones. One of the stable clones, SaOS-2-ER, expressed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of ER mRNA by RT-PCR, and contained an increased level of ER cytosolic protein as detected by an ER-specific EIA. The overexpressed ER was functional and sensitive to E2 in a dose-dependent fashion after transient transfection with a vector containing an estrogen response element (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Scatchard analysis revealed a single high-affinity binding site with a Kd similar to values obtained for the ER in MCF-7 breast cancer cells. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase activity, and decreased IL-6 expression and secretion. In response to E2, a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may provide a sensitive and unique model for understanding the mechanism of E2 and ER in overall bone metabolism.

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines.

The estrogen receptor (ER) mediates the effects of its cognate ligand on important cellular processes such as development of female secondary sexual characteristics, establishment and maintenance of pregnancy, progression of breast cancer, and maintenance of bone mass. We have previously demonstrated that the human ER (hER) gene is transcribed from two promoters, suggesting that tissue- and cell-specific expression patterns of this gene may, at least in part, be regulated by differential promoter usage. Here we show, by using a reverse transcriptase coupled polymerase chain reaction assay, that transcripts initiated from both hER gene promoters were expressed in breast and uterus. In contrast, only transcripts originating from the distal promoter could be detected in human primary osteoblasts. Furthermore, determination of the expression levels of the two hER transcripts by quantitative polymerase chain reaction demonstrated an almost 30-fold increase of the transcripts originating from the proximal promoter in breast cancer cell lines over that detected in normal breast tissue. Taken together, our results demonstrate a previously unrecognized mechanism for regulation of hER gene expression by tissue-specific differential promoter utilization. In addition, our results suggest that estrogen-dependent cell transformation may be accompanied by a change in the relative expression levels of the two hER transcripts.

Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor

Estrogen antagonists such as ICI164,384 do not inhibit 17 beta-estradiol (E2)-dependent gene activity in yeast expressing the mammalian estrogen receptor although these compounds bind to receptors isolated from these cells. Various explanations have been offered for antiestrogen resistance in yeast systems including differences in cell-specific components and lack of permeability of the yeast cell wall to these compounds. We have used a strain of Saccharomyces cerevisiae transformed with the human estrogen receptor gene, and two estrogen response elements linked to a lacZ reporter gene to study the pharmacology of estrogen agonists and antagonists. The rank order of potency of estrogen agonists in this strain (CY525) is similar to that in estrogen-dependent mammalian cells: DES > or = E2 > E1 > E3 = zeranol. Competitive binding with 3H-E2 by these compounds in cell-free extracts of CY525 results in a similar order of potency with a reverse order for E1 and E3. The pure estrogen antagonist ICI164,384 also binds to the receptor from cell-free extracts of CY525 with an IC50 of approximately 14nM. As in mammalian cells ICI164,384 does not induce E2-dependent gene activity. However, unlike mammalian cells, E2-induced gene activity in CY525 is not inhibited by ICI164,384. Intact CY525 cells incubated with 3H-17 beta estradiol were found to specifically bind the labeled ligand since excess unlabeled E2 effectively competed for binding. Unlabeled DES and E1 were also found to compete, however, excess unlabeled ICI164,384, E3 and the second generation antagonist ICI182,720 were unable to displace 3H-E2 binding in intact cells. These results indicate that certain compounds enter the intact yeast cell more readily than others and offer an explanation for antagonist resistance in these organisms.

Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor.

Estrogen antagonists such as ICI164,384 do not inhibit 17 beta-estradiol (E2)-dependent gene activity in yeast expressing the mammalian estrogen receptor although these compounds bind to receptors isolated from these cells. Various explanations have been offered for antiestrogen resistance in yeast systems including differences in cell-specific components and lack of permeability of the yeast cell wall to these compounds. We have used a strain of Saccharomyces cerevisiae transformed with the human estrogen receptor gene, and two estrogen response elements linked to a lacZ reporter gene to study the pharmacology of estrogen agonists and antagonists. The rank order of potency of estrogen agonists in this strain (CY525) is similar to that in estrogen-dependent mammalian cells: DES > or = E2 > E1 > E3 = zeranol. Competitive binding with 3H-E2 by these compounds in cell-free extracts of CY525 results in a similar order of potency with a reverse order for E1 and E3. The pure estrogen antagonist ICI164,384 also binds to the receptor from cell-free extracts of CY525 with an IC50 of approximately 14nM. As in mammalian cells ICI164,384 does not induce E2-dependent gene activity. However, unlike mammalian cells, E2-induced gene activity in CY525 is not inhibited by ICI164,384. Intact CY525 cells incubated with 3H-17 beta estradiol were found to specifically bind the labeled ligand since excess unlabeled E2 effectively competed for binding. Unlabeled DES and E1 were also found to compete, however, excess unlabeled ICI164,384, E3 and the second generation antagonist ICI182,720 were unable to displace 3H-E2 binding in intact cells. These results indicate that certain compounds enter the intact yeast cell more readily than others and offer an explanation for antagonist resistance in these organisms.

Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.

Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene.

Estrogen receptor and its ligand, estradiol, have long been thought to be essential for survival, fertility, and female sexual differentiation and development. Consistent with this proposed crucial role, no human estrogen receptor gene mutations are known, unlike the androgen receptor, where many loss of function mutations have been found. We have generated mutant mice lacking responsiveness to estradiol by disrupting the estrogen receptor gene by gene targeting. Both male and female animals survive to adulthood with normal gross external phenotypes. Females are infertile; males have a decreased fertility. Females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. In adult wild-type and heterozygous females, 3-day estradiol treatment at 40 micrograms/kg stimulates a 3- to 4-fold increase in uterine wet weight and alters vaginal cornification, but the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption. Prenatal male and female reproductive tract development can therefore occur in the absence of estradiol receptor-mediated responsiveness.

Localization of DNase I hypersensitive sites in the human oestrogen receptor gene correlates with the transcriptional activity of two differentially used promoters.

The human oestrogen receptor (hER) gene has recently been shown to be transcribed from two different promoters. In order to study the hER gene promoter region we initially isolated and sequenced 3.3 kb of the 5' region of the hER gene. DNase I hypersensitivity experiments with nuclei from the ER-expressing cell line MCF-7 mapped three different hypersensitive sites, located in the vicinity of the two hER promoters previously identified. However, in another ER-positive cell line, ZR-75-1, DNase I hypersensitivity was observed only in the region of the downstream promoter, suggesting differences between the two cell lines in promoter usage. Polymerase chain reaction-mediated detection of mRNAs transcribed from the two different promoters demonstrated transcripts from the downstream promoter in both the cell lines studied. However, mRNAs transcribed from the upstream promoter could only be detected in MCF-7 cells. Thus, the pattern of DNase I hypersensitivity correlates with the transcriptional activity of the two promoters in the hER gene. Based on these data, we suggest that differential promoter usage might be a novel level of regulation of the hER gene.

Analysis of a DNA sequence upstream of the human estrogen receptor gene.

Analysis of a DNA sequence upstream of the human estrogen receptor gene.

Identification of a functional role for the 3′ region of the human oestrogen receptor gene

A well-conserved feature of the steroid receptor gene family is the presence of an exceptionally long 3' untranslated region (UTR). Analysis of this sequence from the human oestrogen receptor (hER) gene showed the presence of a number of AT-rich regions that included thirteen repeats of the ATTTA motif, an element known to have a destabilizing effect in other systems. In the region 3' of the gene there were a further eight copies of this pentamer. Also located in this sequence were two members of the Alu repetitive family in inverse orientation and in a tandem arrangement. Transfection experiments in which the 3' UTR and 3' flanking sequence were included in chloramphenicol acetyltransferase expression vectors revealed a large destabilization effect with several different fragments. This inherent instability appears to be determined by the primary nucleotide sequence but may act in conjunction with other factors. This post-transcriptional regulatory mechanism may contribute to the control of the level of the hER mRNA.

Organization of a rainbow trout estrogen receptor gene

The complete coding region of the estrogen receptor gene was isolated from a rainbow trout genomic library. This gene is divided into ten exons spanning at least 30 kb of genomic DNA. With two exceptions, intron positions are identical to those of the human estrogen receptor gene. The 5′ end of the gene, including 1.7 kb of the promoter region, was sequenced. This region exhibits several putative regulatory elements. Localization of a potential estrogen responsive element to the first exon suggests that this gene is autoregulated. This 5′ end region was also shown to be able to drive the expression of a CAT reporter gene in Xenopus laevis oocytes.

An estrogen receptor genetic polymorphism and a history of spontaneous abortion--correlation in women with estrogen receptor positive breast cancer but not in women with estrogen receptor negative breast cancer or in women without cancer.

We previously identified a polymorphism in the human estrogen receptor gene. In a preliminary study on women with estrogen receptor positive (ER+) breast tumors, we found that the presence of the rarer of the two alleles, the B' allele, is correlated with a history of spontaneous abortion. Because that study evaluated only women with estrogen receptor positive (ER+) breast cancer, it was unknown whether the observed correlation was restricted to the cancer group or was independent of breast cancer. We have now extended our analysis to include not only additional women with ER+ breast cancer, but also those with estrogen receptor negative (ER-) breast cancer and women without cancer. Results of the current study continue to show an association between the B' allele and a history of spontaneous abortion in the ER+ breast cancer group. There was no such correlation either in the ER- breast cancer group or in the group without cancer. Also, we continue to observe, in the ER+ breast cancer group, a significantly higher concentration of ER protein in tumors from homozygous wild type women (genotype BB), than in the tumors from women who are heterozygous for the rarer allele (genotype BB'). We conclude that the combination of spontaneous abortion and the BB' ER genotype may be a marker for breast cancer susceptibility.

Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists

An expression system that utilized yeast copper metallothionine promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter β-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.

Analysis of upstream sequences of the human estrogen receptor gene

An upstream region of the human estrogen receptor gene (ER) spanning from -2776 to -38 in relation to a previously described exon 1 was sequenced and analyzed for the possible presence of open reading frames (ORF) and regulatory motifs. We identified a long 459 bp ORF beginning at -2301. Most of the transcriptional regulatory motifs present within the ER upstream sequence are distributed either upstream or downstream of this long ORF which displays a TATA box, a CACC motif and a canonical cap site in the 5' flanking region.