Potential Estrogen Response Element Research Articles

Estradiol regulates the expression of CD45 splicing isoforms in lymphocytes.

CD45, a common leukocyte antigen expressed on the surface of all nucleated hematopoietic cells, indicates the developmental stage and functional status of lymphocytes by its alternative splicing isoforms. Estrogen is correlated with the immune activity of lymphocytes and is involved in the sex bias of several human autoimmune diseases, but the effect of estrogen on the expression of the CD45 splicing isoforms remains unknown. In the present study, a potential estrogen response element was identified on the opposite strand of the CD45 gene by bioinformatics software prediction. The results from RT-qPCR results showed that the expression levels of CD45RO isoform and CD45 antisense RNA were increased after the lymphocytes were treated with 10nM 17beta-estradiol, and this effect of 17beta-estradiol was reversed when the lymphocytes were cotreated with an estrogen receptor antagonist. Moreover, bisulfite sequencing PCR showed that CD45 DNA methylation in lymphocytes was increased after the treatment with 10nM 17beta-estradiol. In conclusion, estradiol regulated the expression of CD45 in an estrogen receptor-dependent manner and was associated with CD45 antisense RNA and DNA methylation. The results helped elucidate the regulatory mechanism of the expression of CD45 isoforms and the correlation between estrogen levels and immune activity in females.

Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of cystatin A in breast cancer cells

Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17β-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

Abstract 1851: E6-associated protein-dependent estrogen receptor modulation of protein kinase A regulatory subunit R2A expression in neuroblastoma

Abstract Purpose/Objective(s): Steroid hormone receptors, upon hormonal activation, act to modulate gene expression in receptive cells through interactions with coregulator proteins. Protein Kinase A (PKA) is a particularly potent downstream effector, and is conservatively regulated in a number of biochemical pathways. The current project aims to elucidate the mechanisms of action of E6AP, a known critical player in endocrine cancer on the expression/activity of PKA in neuroblastoma. Materials/Methods: A microarray genetic analysis yielded a decreased PKA-R2A expression dependent upon decreased levels of estradiol (E2) or E6AP. This correlation was validated via a PCR determined downregulation of PKA-R2A mRNA with E6AP knockdown in Neuro2a cells. Bioinformatics provided four potential Estrogen Response Element (ERE) sequences in the PKA-R2A promoter region. These sequences were cloned to a Luciferase reporter gene vector. HeLa cells harboring these constructs were incubated with and without E2. Transcriptional activity was surrogated by bioluminescence via a Luciferase-Luciferin reaction, normalized to Renilla luminal output. Electrophoretic mobility shift assay (EMSA) was performed to detect direct ER binding to biotin-labeled ERE in the presence and absence of E2. ER-specific antibodies generated a super-shift. Unlabeled and consensus ERE allowed for competitive binding. Chromatin immunoprecipitation (ChIP) investigated recruitment of the complete transcriptional complex E6AP-ER onto the promoter region of the PKA-R2A gene and its association with active expression of the gene in Neuro2a cells. Results: A Luciferase luminal output to unit Renilla luminal output ratio was obtained in the absence and presence of E2. The scrambled control yielded ratios of 2.77 and 1.97, respectively. The consensus control yielded 9.55 and 85.37. ERE-1 yielded significant ratios of 8.57 and 55.7. EMSA evaluation of ERE-1 demonstrated a biotin-labeled ERE band shift in the presence of ER incubated with E2 relative to that without E2, and a super-shift with addition of an Anti-ER antibody. Competitive saturation with both the unlabeled and consensus ERE sequences negated these observed shifts. ChIP results show, upon E2 treatment in Neuro2a cells, increased recruitment of the E6AP-ER complex to the same ERE-1 associated with increased H3K14 acetylation and p300 recruitment at the same site which was coupled with the recruitment of pRNAPolII to the transcriptional start site of the PKA-R2A gene indicating active transcription and expression of the gene. Conclusion: ERE sequences exist and appear to function in response to E2 within the PKA-R2A promoter region, modulating the transcriptional status of a ubiquitous and crucial protein. Novel therapies targeting this hormonal foundation may potentially play a role in ameliorating the symptomatic manifestations associated with neuroblastoma. Citation Format: Jean-Pierre Obeid, Nawal Zafar, Jimmy El Hokayem. E6-associated protein-dependent estrogen receptor modulation of protein kinase A regulatory subunit R2A expression in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1851.

Transcriptional regulation of the sodium-coupled neutral amino acid transporter (SNAT2) by 17β-estradiol.

The sodium-coupled neutral amino acid transporter 2 (SNAT2) translocates small neutral amino acids into the mammary gland to promote cell proliferation during gestation. It is known that SNAT2 expression increases during pregnancy, and in vitro studies indicate that this transporter is induced by 17β-estradiol. In this study, we elucidated the mechanism by which 17β-estradiol regulates the transcription of SNAT2. In silico analysis revealed the presence of a potential estrogen response element (ERE) in the SNAT2 promoter. Reporter assays showed an increase in SNAT2 promoter activity when cotransfected with estrogen receptor alpha (ER-α) after 17β-estradiol stimulation. Deletion of the ERE reduced estradiol-induced promoter activity by 63%. Additionally, EMSAs and supershift assays showed that ER-α binds to the SNAT2 ERE and that this binding competes with the interaction of ER-α with its consensus ERE. An in vivo ChIP assay demonstrated that the binding of ER-α to the SNAT2 promoter gradually increased in the mammary gland during gestation and that maximal binding occurred at the highest 17β-estradiol serum concentration. Liquid chromatography-elevated energy mass spectrometry and Western blot analysis revealed that the SNAT2 ER-α-ERE complex contained poly(ADP-ribose) polymerase 1, Lupus Ku autoantigen protein p70, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) proteins and that the silencing of each of these proteins nearly abolished 17β-estradiol-stimulated SNAT2 promoter activity. Nuclear levels of GAPDH increased progressively during gestation in the mammary gland, and GAPDH binding was nucleotide-specific for the SNAT2 ERE. Thus, this study provides new insights into how the mammary epithelium adapts to control amino acid uptake through the transcriptional regulation of the SNAT2 transporter via 17β-estradiol.

Transcriptional regulation of the sodium‐coupled neutral amino acid transporter (SNAT2) by 17β‐estradiol

The SNAT2 amino acid transporter allows the entry of small neutral amino acids into the mammary gland to promote cell proliferation during gestation and milk protein synthesis during lactation. During pregnancy, 17β‐estradiol promotes the expression of this transporter by increasing SNAT2 promoter activity. The objective of this work was to elucidate the mechanism by which the 17β‐estradiol regulates the transcription of SNAT2. Computational analysis of the promoter region of SNAT2 showed the presence of a potential estrogen response element (ERE). Studies in HeLa cells using the luciferase reporter gene showed that the activity of the promoter region of SNAT2 increased with the co‐transfection of constructions with progressive deletions of SNAT2 promoter, and the plasmid that expresses the estrogen receptor alpha (ERα). The highest luciferase activity was found with the construct containing the ERE (40%), but 20% of activity remained in smaller constructions. EMSA and supershift assays showed that ERα binds to the ERE in SNAT2 promoter and this binding competed with ERα interaction to its consensus sequence. This binding was also demonstrated in vivo by ChIP assay where ERα bound to the promoter region of SNAT2 in the mammary gland of pregnant rats on days 5, 14 and 20 of gestation. We conclude that 17β‐estradiol increased SNAT2 promoter activity in the presence of ERα by a classic genomic pathway.

Transcriptional regulation of bidirectional gene pairs by 17-β-estradiol in MCF-7 breast cancer cells

Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5'flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40% of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17% of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17β-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17β-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.

Matrix metalloproteinase-26 (Matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer

Objective. Matrix metalloproteinases (MMPs) are key players in the degradation of extracellular matrix and basement membranes, and are thus important in tumor invasion. Recently, MMP-26 (endometase), a novel matrilysin-type member of the MMP family, was cloned from an endometrial tumor. This study examines the expression of MMP-26 mRNA in hyperplastic, premalignant and malignant endometrial samples, and compares with normal endometrial tissue. Methods. Endometrial carcinoma samples (19) were histologically classified as well, intermediately and poorly differentiated. Samples with hyperplasia ( n = 15) were classified as simple, complex, or complex with atypia. Normal endometrial specimens ( n = 39) were classified according to an ideal 28-day menstrual cycle and subsequently grouped in the early, middle, and late parts of the cycle. All samples were analyzed using in situ hybridization and real time PCR. The probes used for in situ hybridization and real time PCR recognized non-overlapping sequences. MMP-26 protein was localized by immunohistochemistry. Results. MMP-26 mRNA was exclusively localized in the epithelial component of normal, hyperplastic, premalignant, as well as malignant samples. It was not found in the stroma of any tissue category. Quantifications with real time PCR as well as semi-quantifications of the in situ hybridization signal revealed maximal levels in normal tissue at midcycle and in endometrial hyperplasia both with and without atypia. The amount of MMP-26 mRNA decreased progressively with loss of histological differentiation in malignant samples. Immunostaining localized MMP-26 in epithelial glandular and luminal cells, in vessel walls, and in tumor cells. Since the pattern of MMP-26 expression mimicked that of ER-α, we searched the MMP-26 promoter region for a potential estrogen response element (ERE). A sequence at position −130 to −116 had high homology to the consensus sequence of an ERE. Based on these observations, we suggest that ER-α is involved in regulation of the MMP-26 gene. Conclusions. MMP-26 mRNA is selectively localized in the epithelial compartment of normal, hyperplastic, and malignant endometrial tissue. Expression is high in normal and hyperplastic endometria, but is downregulated in the late part of the cycle and in malignant tumors. The expression pattern of MMP-26 mRNA mimics that of ER-α, and the promoter region of the MMP-26 gene has a potential ERE.

Estrogen receptor-mediated repression of human hepatic lipase gene transcription

Estrogen replacement therapy in women decreases hepatic lipase (HL) activity, which may account for the associated increase in HDL cholesterol. To investigate whether estrogen decreases HL transcription, transient cotransfection assays with HL promoter and estrogen receptor-α (ERα) expression constructs were performed in HepG2 cells. 17β-estradiol (E2) decreased transcription driven by the −1557/+41 human HL promoter by up to 50% at 10−7 M. Mutation of ERα by deletion of its transactivation domains or ligand-binding domain eliminated E2-induced repression of the promoter, whereas deletion of the DNA-binding domain of ERα resulted in a 7-fold activation by E2. The E2-induced repression was maintained after mutation of a potential estrogen-response element in the promoter. The region of estrogen responsiveness was localized to −1557/ −1175 of the HL promoter by deletion analysis. Mutation of an AP-1 site at −1493 resulted in a partial loss of E2-induced repression, similar to that caused by deletion of nucleotides −1557 to −1366. Gel shift assays with nuclear extracts from E2-treated HepG2 cells stably expressing ERα demonstrated an increase in binding to an AP-1 consensus oligonucleotide. The AP-1 activator, phorbol 12-myristate 13-acetate, inhibited the HL promoter by greater than 50%. Collectively, the data suggest that estrogen represses the transcription of the HL gene, possibly through an AP-1 pathway. —Jones, D. R., R. J. Schmidt, R. T. Pickard, P. S. Foxworthy, and P. I. Eacho. Estrogen receptor-mediated repression of human hepatic lipase gene transcription.

Estrogen Modulation of Apolipoprotein(a) Expression: IDENTIFICATION OF A REGULATORY ELEMENT

Elevated plasma levels of the lipoprotein particle Lp(a) are a major risk factor for cardiovascular disease. Lp(a) plasma levels are determined by the level of expression of its characteristic protein component, apo(a). Apo(a) expression is modulated by several hormones, of which estrogens are the best known. The chromosomal region responsible for estrogen response was identified within an apo(a) enhancer located at approximately 26 kilobases from the apo(a) promoter. Although the estrogen-responsive unit contains a potential estrogen response element, binding of estrogen receptor-alpha to DNA was not necessary. The receptor, activated by bound estradiol, interacts through its transactivation domains with a transcription factor necessary for the function of the enhancer, preventing its binding to DNA.

Estrogen receptor does not directly regulate the murine Muc-1 promoter

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17- β-estradiol (E 2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at −748 to −735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ER α nor ER β bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5′ Muc-1 flanking sequences showed that E 2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E 2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ER α, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within −1838 to +43 bp do not respond to E 2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between −73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E 2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.

Pituitary-type transcription of the human prolactin gene in the absence of Pit-1.

We describe a human (h) PRL-producing cell line, SKUT-1B-20, which we isolated as a subclone of a uterine sarcoma cell line. Although this cell line is of uterine origin, it does not use the decidual-specific upstream promoter of the hPRL gene, but transcribes the hPRL gene from the downstream pituitary-type transcription start site, as determined by Northern blot, reverse transcriptase-polymerase chain reaction and primer extension analyses. This is particularly intriguing because SKUT-1B-20 cells lack the transcription factor Pit-1. No Pit-1 messenger RNA was detectable by reverse transcriptase-polymerase chain reaction, and endogenous Pit-1 target genes (GH, PRL, and Pit-1) were refractory to transfected Pit-1 expression vector, whereas in cotransfection experiments, Pit-1 efficiently activated reporter gene fusion constructs carrying 5'-flanking sequences of the human and rat PRL or the mouse Pit-1 genes. By transfecting reporter genes containing 8.7 kilobases of DNA flanking the hPRL pituitary-specific start site (hPRL-8700/Luc) and deletions thereof, we located a Pit-1-independent cis-active region more than 7 kilobases upstream of the start site. The most distal 1650 or 880 base pairs of the hPRL genomic fragment (which extends to -8784 base pairs), when placed directly upstream of the homologous hPRL or the heterologous thymidine kinase promoters, conferred transcriptional activation to those promoters. SKUT-1B-20 cell-specific activation of hPRL-8700/Luc could not be suppressed by the introduction of an inhibitor of protein kinase A (PKA), PKI. This is the first demonstration of pituitary-type PRL gene transcription independent of Pit-1 and activation of the PKA pathway. The SKUT-1B-20 cell line was then used in reconstitution experiments to delineate the role of Pit-1 in modulating the transcriptional effects of phorbol ester, PKA, and estrogen receptor (ER) on the hPRL gene. The low response of hPRL/luciferase fusion genes to phorbol ester was greatly enhanced by cotransfected Pit-1 and was mediated by the proximal region between -250 and -38. The catalytic subunit of PKA, C beta, was able to elicit a moderate induction of hPRL-8700/Luc even in the absence of Pit-1. A potential estrogen response element has been located in the hPRL gene sequence at a position similar to that of the estrogen response element of the rat PRL gene immediately adjacent to the distal enhancer.(ABSTRACT TRUNCATED AT 400 WORDS)