Mesut Muyan

Sequence Requirements for Estrogen Receptor Binding to Estrogen Response Elements

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Single-Chain Estrogen Receptors (ERs) Reveal that the ERα/β Heterodimer Emulates Functions of the ERα Dimer in Genomic Estrogen Signaling Pathways

ABSTRACT The effects of estrogens, particularly 17β-estradiol (E2), are mediated by estrogen receptor α (ERα) and ERβ. Upon binding to E2, ERs homo- and heterodimerize when coexpressed. The ER dimer then regulates the transcription of target genes through estrogen responsive element (ERE)-dependent and -independent pathways that constitute genomic estrogen signaling. Although ERα and ERβ have similar ERE and E2 binding properties, they display different transregulatory capacities in both ERE-dependent and -independent signaling pathways. It is therefore likely that the heterodimerization provides novel functions to ERs by combining distinct properties of the contributing partners. The elucidation of the role of the ER heterodimer is critical for the understanding of physiology and pathophysiology of E2 signaling. However, differentially determining target gene responses during cosynthesis of ER subtypes is difficult, since dimers formed are a heterogeneous population of homo- and heterodimers. To circumvent the pivotal dimerization step in ER action and hence produce a homogeneous ER heterodimer population, we utilized a genetic fusion strategy. We joined the cDNAs of ERα and/or ERβ to produce single-chain ERs to simulate the ER homo- and heterodimers. The fusion ERs interacted with ERE and E2 in a manner similar to that observed with the ER dimers. The homofusion receptors mimicked the functions of the parent ER dimers in the ERE-dependent and -independent pathways in transfected mammalian cells, whereas heterofusion receptors emulated the transregulatory properties of the ERα dimer. These results suggest that ERα is the functionally dominant partner in the ERα/β heterodimer.

CCDC62/ERAP75 functions as a coactivator to enhance estrogen receptor beta-mediated transactivation and target gene expression in prostate cancer cells

Human prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER) beta, but not ERalpha. ERbeta might utilize various ER coregulators to mediate the E2-signaling pathway in PCa. Here, we identified coiled-coil domain containing 62 (CCDC62)/ERAP75 as a novel ER coactivator. CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, glutathione S-transferase pull-down and coimmunoprecipitation methods proved that ERbeta can interact with the C-terminus of CCDC62/ERAP75 via the ligand-binding domain. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ERbeta and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP75 can be recruited by the estrogen response element-ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ERAP75 within the promoter of the estrogen-responsive gene cyclin D1. In addition, using silencing RNA (siRNA) against endogeneous CCDC62/ERAP75, we demonstrated that inhibition of endogenous CCDC62/ERAP75 results in the suppression of ERbeta-mediated transactivation as well as target gene expression in LNCaP cells. More importantly, using the tet-on overexpression system, we showed that induced expression of CCDC62/ERAP75 can enhance the E2-regulated cyclin D1 expression and cell growth in LNCaP cells. Together, our results revealed the role of CCDC62/ERAP75 as a novel coactivator in PCa cells that can modulate ERbeta transactivation and receptor function.

Targeting estrogen responsive elements (EREs): design of potent transactivators for ERE-containing genes.

The estrogen hormone (E2) plays an important role in the physiology and pathophysiology of target tissues. The effects of E2 are conveyed by the estrogen receptors (ER) alpha and beta. The E2-ER complex mediates an array of genomic and non-genomic events that orchestrate the expression of a number of genes involved in the regulation of cell proliferation and differentiation. The interaction of with the regulatory DNA sequence, estrogen responsive element (ERE), of each responsive gene constitutes a critical genomic signaling pathway. However, the relative importance of ERE-dependent E2-ER signaling in cell proliferation remains to be elucidated. To address this issue, we engineered ERE-binding activators (EBAs) that specifically and potently regulate ERE-containing genes. The modular nature of ER allowed us to initially design a monomeric ERE-binding module by genetically joining two DNA-binding domains with the hinge domain. Integration of strong activation domains from other transcription factors into this module generated constitutively active EBAs. These transactivators robustly induced the expression of only ERE-containing promoter constructs in transfected cells independent of ligand, dimerization, ER-subtype and -status. Moreover, EBAs altered cell cycle progression in breast cancer cell lines in a manner similar to E2-ER. These results demonstrate the importance of ERE-containing genes in the regulation of cell proliferation. These novel ERE-binding transregulators could also be a basis for the targeted regulation of ERE-containing genes, the identification of estrogen responsive gene networks, and the development of alternative/complementary therapeutic approaches for estrogen target tissue cancers.

Molecular Basis of Therapeutic Strategies for Breast Cancer

The development of breast cancer is the consequence of uncontrolled growth and division of breast-ductal epithelial cells. While many factors contribute to its etiology, estrogen hormones within the context of many interrelated growth signaling pathways play critical roles for the initiation and development of breast cancer. The effects of estrogens are primarily mediated by the estrogen receptors (ERs) alpha and beta. ER mediates a complex array of genomic and non-genomic events that orchestrate cellular metabolism, mitogenesis, morphogenesis, motogenesis, and apoptosis. The current modalities for the treatment of breast cancer have centered on the development of agents with diverse pharmacology to reduce/ablate the circulating estrogens or to alter/prevent ER function. Approaches to perturb the estrogen environment are successful usually in the remission of established tumors. However, many breast tumors are not responsive or eventually develop resistance to endocrine therapies. Despite considerable effort, the mechanism for the non-responsiveness and acquisition of resistance remains unclear. The establishment of hormone responsiveness is one of the current approaches for the development of an effective therapeutic modality for de novo resistant breast tumors. Re-establishment of loss of ER synthesis/function, on the other hand, constitutes a primary therapeutic goal for acquired resistance neoplasms. We have recently engineered transregulatory proteins that specifically targeted and robustly regulated estrogen responsive genes independent of ligand, ER-subtype and cell-context. The targeted regulation of estrogen responsive gene networks by these designer transregulators could provide a basis for the development of novel approaches for experimental biology and medicine.

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor α Are Required to Elicit Cellular Alterations

Estrogen (E2) signaling is conveyed by the transcription factors estrogen receptor (ER) α and β. ERs modulate the expression of genes involved in cellular proliferation, motility, and death. The regulation of transcription by E2-ERα through binding to estrogen-responsive elements (EREs) in DNA constitutes the ERE-dependent signaling pathway. E2-ERα also modulates gene expression by interacting with transregulators bound to cognate DNA-regulatory elements, and this regulation is referred to as the ERE-independent signaling pathway. The relative importance of the ERE-independent pathway in E2-ERα signaling is unclear. To address this issue, we engineered an ERE-binding defective ERα mutant (ERαEBD) by changing residues in an α-helix of the protein involved in DNA binding to render the receptor functional only through the ERE-independent signaling pathway. Using recombinant adenovirus-infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that E2-ERαEBD modulated the expression of a subset of ERα-responsive genes identified by microarrays and verified by quantitative PCR. However, E2-ERαEBD did not affect cell cycle progression, cellular growth, death, or motility in contrast to E2-ERα.ERαEBD in the presence of E2 was also ineffective in inducing phenotypic alterations in ER-negative U-2OS cells derived from an osteosarcoma. E2-ERα, on the other hand, effectively repressed growth in this cell line. Our findings suggest that genomic responses from the ERE-dependent signaling pathway are required for E2-ERα to induce alterations in cellular responses.

ERAP75 functions as a coactivator to enhance estrogen receptor α transactivation in prostate stromal cells

Estrogen receptor α (ERα) has been reported to be expressed and function in the prostate stromal cells, and numerous evidences indicated that the stromal ERα signal pathway plays critical roles in prostate development and cancer. ERα requires distinct coregulators for efficient transcriptional regulation. The goal of this study is to examine physical and functional interaction between ERα and ERAP75 in the context of prostate stromal cells.

Do Estrogen Receptor β Polymorphisms Play A Role in the Pharmacogenetics of Estrogen Signaling

Estrogen hormones play critical roles in the regulation of many tissue functions. The effects of estrogens are primarily mediated by the estrogen receptors (ER) alpha and beta. ERs are ligand-activated transcription factors that regulate a complex array of genomic events that orchestrate cellular growth, differentiation and death. Although many factors contribute to their etiology, estrogens are thought to be the primary agents for the development and/or progression of target tissue malignancies. Many of the current modalities for the treatment of estrogen target tissue malignancies are based on agents with diverse pharmacology that alter or prevent ER functions by acting as estrogen competitors. Although these compounds have been successfully used in clinical settings, the efficacy of treatment shows variability. An increasing body of evidence implicates ERalpha polymorphisms as one of the contributory factors for differential responses to estrogen competitors. This review aims to highlight the recent findings on polymorphisms of the lately identified ERbeta in order to provide a functional perspective with potential pharmacogenomic implications.

Estrogen receptors similarly mediate the effects of 17β-estradiol on cellular responses but differ in their potencies

Abstract17β-estradiol (E2), as the main circulating estrogen hormone, plays critical roles in the physiology and pathophysiology of various tissues. The E2 information is primarily conveyed by the transcription factors, estrogen receptors (ERs) α and β. ERs share similar structural and functional features. Experimental studies indicate that upon binding to E2, ERs directly or indirectly interact with DNA and regulate gene expressions with ERα being more potent transregulator than ERβ. However, studies also showed that ERβ induces alterations in phenotypic features of cancer cell lines independent of E2. These observations suggested that the manner in which the unliganded ERβ induces phenotypic alterations in cancer cell models differs from that of ERα. Studies demonstrated that while requiring E2 for function at low levels of synthesis, the unliganded ERα at augmented concentrations modulates gene expressions and cellular growth. We, therefore, anticipated that heightened levels of ERβ synthesis could similarly circumvent the dependency on E2 leading to gene transcriptions and cellular proliferation. To test this prediction, we used adenovirus-infected cancer cell lines in which ERs were shown to induce genomic and cellular responses. We found that while ERβ at low levels of synthesis was dependent upon E2 for function, the receptor at high levels regulated gene expression and cellular proliferation independent of E2. We then addressed whether ERs at comparable levels that require E2 for function differentially alter gene expressions and cellular responses. We found that ERs mediate the effects of E2 on gene expression, cellular proliferation, apoptosis, and motility with an overlapping pattern. However, ERα was more potent regulator than ERβ in inducing cellular responses. Our results suggest that differences in potencies to regulate the expression of genes are a critical feature of the ER subtypes in mediating E2 signaling in cancer cell lines.