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Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

Background: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments., Method: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts., Results: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]., Conclusions: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Constitutively active estrogen receptor α (ERα) variants with C-terminal truncation are candidate molecules for gain of both endocrine- and chemotherapy-resistance in estrogen-sensitive tumors. Our previous reports using artificially truncated ERα constructs demonstrated that ERα variants encoded in 1-2-3-cryptic exon- and 1-2-3-4-cryptic exon-types of transcripts have potentials to display constitutive transactivation of an estrogen response element reporter. However, naturally occurring 1-2-3-cryptic exon-type ERα variants have not been cloned yet. Therefore, the present study was designed to identify naturally occurring ERα variants encoded in 1-2-3-cryptic exon-type ERα transcripts. We cloned a novel C-terminally truncated ERα variant (ERαi34) encoded in a 1-2-3-i34 transcript from MCF-7 cells and characterized its constitutive and ER antagonist-resistant transactivation in transfected cells. Stable transfection of the variant into MCF-7 cells augmented basal cell proliferation insensitive to fulvestrant. Collectively, we validated the structure-based mechanisms underlying constitutive and ER antagonist-resistant transactivation by C-terminally truncated ERα variants., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflicts of interest to disclose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The estrogen receptor α (ERα) directs transactivation of target genes, and splice variants have been shown to exhibit altered activation properties. We previously documented the complicated alternative promoter usage and splicing patterns of the rat ERα gene; however, the information was restricted to a few specific organs. Therefore, we re-examined the rat mRNA profiles of ERα, including the generation of the exon 1-skipping, ERα46 transcript in a wider variety of rat organs and further characterized the fundamental functional properties of rat ERα46 variants. With the use of RT-PCR, we discovered unique distribution and splicing patterns for promoter-specific ERα isoforms, as well as the extensive expression of the Δ exon 1 variant in the rat. Similar to wild-type ERα, an immunocytochemical analysis showed a predominant localization of ERα46 proteins in the nuclei of transfected cells. Luciferase reporter assays revealed that ERα46 variants stimulated the transcriptional activity of an estrogen response element-driven promoter in response to estrogen. In addition, the variants exhibited distinct transactivation and reactivity to 4-hydroxytamoxifen in different cell types. Although the alternative splicing patterns are species-specific, the profiles of the alternative use of promoters, and the fundamental properties of the rat ERα46 variant are similar to those of human and mouse homologs. Therefore, the present study provides fundamental and useful information for further research into the regulation and functions of ERα gene variants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Expression of the estrogen receptor α (ERα) gene is subject to complex regulation. To elucidate the mechanisms of this regulation, the genomic organization and the physiological role of the multiple 5'-untranslated regions (5'-UTRs) must be determined. Here, we investigated the expression and splicing patterns of the human ERα E isoforms. We identified two novel untranslated exons, N1 and N2, in the 5'-region of the human ERα gene and multiple E isoform mRNA variants generated by alternative usage of non-coding internal exons. Expression of the N1-containing variants was observed only in the human breast adenocarcinoma cell line, MCF7, while the N2-containing variants were expressed in the adult liver and MCF7 cells. We examined post-transcriptional regulation of the variant mRNAs using luciferase reporter assays and quantitative PCR. The insertion of untranslated internal exons into the 5'-UTRs of the E isoforms reduced their translation efficiency, but barely influenced mRNA turnover. Our results indicate that the genomic organization of the human ERα gene and the splicing profiles of the human ERα E isoforms are more complicated than previously reported. Furthermore, the 5'-UTRs of the E isoforms post-transcriptionally control human ERα expression mainly through translational repression., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The 5'-untranslated region (5'-UTR) of the estrogen receptor α (ERα) gene plays an important role in determining its tissue-specific expression. We examined the 5'-UTRs of the mouse ERα mRNA variants in depth using the Basic Local Alignment Search Tool (BLAST), rapid amplification of 5'-cDNA ends (5'-RACE) and RT-PCR. We demonstrated the presence of multiple variants containing unique 5'-UTRs. We mapped the cDNA sequences onto the mouse genome, and found that both alternative splicing from four different leader exons (A, C, F1, and H) to exon 1, and combinations of 12 internal exons (X1, X2, X3, X4, F2/X5, X6, X7, X8, X9, X10, X11, and B) generate multiple ERα transcripts. Mouse exon B, that has homologies with human exon B and rat exon 0T, was used as an internal exon, not as a leader exon. RT-PCR analysis revealed distinct expression patterns of the variants, suggesting that the alternative promoter usage and alternative splicing are regulated in a tissue-specific manner. Our results indicate that the genomic organization of the mouse ERα gene is complicated as previously shown in the rat ERα gene. In addition, both alternative promoter usage and alternative splicing contribute to the remarkable mRNA diversity of the mouse ERα gene., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5'- or 3'-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3'-terminal and 5'-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10 nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The 5'-untranslated regions (5'-UTRs) of the estrogen receptor α (ERα) mRNAs play important roles in the modulation of translation. To elucidate the mechanisms regulating human ERα gene expression, it is necessary to determine its genomic organization and the roles of the multiple 5'-UTRs. We therefore examined the splicing and expression profiles of the human ERα F isoforms. A novel non-coding exon E3 was found in the 5'-region of the human ERα gene and six F isoform mRNA variants were identified. The variants revealed the preferred expression patterns. Post-transcriptional regulation was also investigated. The 5'-UTRs of the F isoforms decreased the translation efficiency but had no effect on mRNA stability. The results indicate that the organization and splicing patterns of the human ERα gene are more complex than previously reported, and that the 5'-UTRs of the F isoforms contribute to the post-transcriptional control of human ERα gene expression through translation repression., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The 5'-untranslated region (UTR) of the estrogen receptor alpha (ERalpha) gene plays an important role in determining tissue-specific expression. To elucidate the regulatory mechanisms of rat ERalpha gene expression, the genomic organization must be investigated. We therefore analyzed the structure of the rat ERalpha mRNA 5'-UTR using rapid amplification of 5'-cDNA ends (5'-RACE) and RT-PCR. The analysis showed the presence of multiple variants containing unique 5'-UTRs. We mapped the cDNA sequences on the rat genome, and newly identified one leader exon (exon 0U) and ten untranslated internal exons (exons I1-10). Both splicing from four different leader exons (exons 0S, 0N, 0U, and 0/B) onto exon 1 and alternative splicing in combination with eleven internal exons (exons I1-10, and 0T) produce multiple transcripts. RT-PCR analysis revealed that each variant had preferred expression sites, suggesting that promoter usage and splicing are regulated in tissue-specific manners. Moreover, we determined a splicing event to yield Deltaexon 1 variants (0S-2-3-4-5-6-7-8), which are translated into rat 46 kDa ERalpha proteins. Our results indicate that the rat ERalpha gene is more complex than previously thought in terms of genomic organization and that both alternative promoter usage and alternative splicing contribute to the remarkable diversity of ERalpha mRNAs., (Copyright 2009 Elsevier Ltd. All rights reserved.)