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MOTIVATION: Next generation sequencing enabled the fast accumulation of genomic data at public repositories. This technology also made it possible to better understand the regulation of gene expression by transcription factors (TFs) and various chromatin-associated proteins through the integration of chromatin immunoprecipitation (ChIP-Seq). The Cistrome Project has become one of the indispensable research portals for biologists to access and analyze data generated with thousands of ChIP-Seq experiments. Integrative motif analysis on shared binding regions among a set of experiments is not yet achievable despite a set of search and analysis tools provided by Cistrome via its web interface and the Galaxy framework. RESULTS: We implemented a python command-line tool for searching binding sequences of a TF common to multiple ChIP-Seq experiments. We use the peaks in the Cistrome database as identified by MACS 2.0 for each experiment and identify shared peak regions in a genomic locus of interest. We then scan these regions for binding sequences using a binding motif of a TF obtained from the JASPAR database. MotifGenie is developed in collaboration with molecular biologists and its findings are corroborated by laboratory experiments. AVAILABILITY AND IMPLEMENTATION: MotifGenie is freely available at https://github.com/ceragoguztuzun/MotifGenie.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição , Análise de Sequência de DNA , Sítios de Ligação/genética , Imunoprecipitação da Cromatina , Fatores de Transcrição/metabolismoRESUMO
17ß-Estradiol (E2), as the main circulating estrogen hormone, regulates many tissue and organ functions in physiology. The effects of E2 on cells are mediated by the transcription factors and estrogen receptor (ER)α and ERß that are encoded by distinct genes. Localized at the peri-membrane, mitochondria, and the nucleus of cells that are dependent on estrogen target tissues, the ERs share similar, as well as distinct, regulatory potentials. Different intracellular localizations of the ERs result in dynamically integrated and finely tuned E2 signaling cascades that orchestrate cellular growth, differentiation, and death. The deregulation of E2-ER signaling plays a critical role in the initiation and progression of target tissue malignancies. A better understanding of the complex regulatory mechanisms that underlie ER actions in response to E2 therefore holds a critical trajectory for the development of novel prognostic and therapeutic approaches with substantial impacts on the systemic management of target tissue diseases.
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YPEL2 is a member of the evolutionarily conserved YPEL family involved in cellular proliferation, mobility, differentiation, senescence, and death. However, the mechanism by which YPEL2, or YPEL proteins, mediates its effects is largely unknown. Proteins perform their functions in a network of proteins whose identities, amounts, and compositions change spatiotemporally in a lineage-specific manner in response to internal and external stimuli. Here, we explored interaction partners of YPEL2 by using dynamic TurboID-coupled mass spectrometry analyses to infer a function for the protein. Our results using inducible transgene expressions in COS7 cells indicate that proximity interaction partners of YPEL2 are mainly involved in RNA and mRNA metabolic processes, ribonucleoprotein complex biogenesis, regulation of gene silencing by miRNA, and cellular responses to stress. We showed that YPEL2 interacts with the RNA-binding protein ELAVL1 and the selective autophagy receptor SQSTM1. We also found that YPEL2 localizes stress granules in response to sodium arsenite, an oxidative stress inducer, which suggests that YPEL2 participates in stress granule-related processes. Establishing a point of departure in the delineation of structural/functional features of YPEL2, our results suggest that YPEL2 may be involved in stress surveillance mechanisms.
Assuntos
Estresse Oxidativo , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The vicinal diaryl heterocyclic framework has been widely used for the development of compounds with significant bioactivities. In this study, a series of diaryl heterocycles were designed and synthesized based on an in-house diaryl isoxazole derivative (9), and most of the newly synthesized derivatives demonstrated moderate to good antiproliferative activities against a panel of hepatocellular carcinoma and breast cancer cells, exemplified with the diaryl isoxazole 11 and the diaryl pyrazole 85 with IC50 values in the range of 0.7-9.5 µM. Treatments with both 11 and 85 induced apoptosis in these tumor cells, and they displayed antitumor activity in vivo in the Mahlavu hepatocellular carcinoma and the MDA-MB-231 breast cancer xenograft models, indicating that these compounds could be considered as leads for further development of antitumor agents. Important structural features of this compound class for the antitumor activity have also been proposed, which warrant further exploration to guide the design of new and more potent diaryl heterocycles.
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CXXC5 is a member of the zinc-finger CXXC family proteins that interact with unmodified CpG dinucleotides through a conserved ZF-CXXC domain. CXXC5 is involved in the modulation of gene expressions that lead to alterations in diverse cellular events. However, the underlying mechanism of CXXC5-modulated gene expressions remains unclear. Proteins perform their functions in a network of proteins whose identities and amounts change spatiotemporally in response to various stimuli in a lineage-specific manner. Since CXXC5 lacks an intrinsic transcription regulatory function or enzymatic activity but is a DNA binder, CXXC5 by interacting with proteins could act as a scaffold to establish a chromatin state restrictive or permissive for transcription. To initially address this, we utilized the proximity-dependent biotinylation approach. Proximity interaction partners of CXXC5 include DNA and chromatin modifiers, transcription factors/co-regulators, and RNA processors. Of these, CXXC5 through its CXXC domain interacted with EMD, MAZ, and MeCP2. Furthermore, an interplay between CXXC5 and MeCP2 was critical for a subset of CXXC5 target gene expressions. It appears that CXXC5 may act as a nucleation factor in modulating gene expressions. Providing a prelude for CXXC5 actions, our results could also contribute to a better understanding of CXXC5-mediated cellular processes in physiology and pathophysiology.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5' untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5'-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G-C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.
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Ilhas de CpG , Proteínas de Ligação a DNA , Fatores de Transcrição , Metilação de DNA , Histonas/metabolismo , Transdução de Sinais , Dedos de ZincoRESUMO
The main circulating estrogen hormone 17beta-estradiol (E2) contributes to the initiation and progression of breast cancer. Estrogen receptors (ERs), as transcription factors, mediate the effects of E2. Ablation of the circulating E2 and/or prevention of ER functions constitute approaches for ER-positive breast cancer treatments. These modalities are, however, ineffective in de novo endocrine-resistant breast neoplasms that do not express ERs. The interaction of E2-ERs with specific DNA sequences, estrogen responsive elements (EREs), of genes constitutes one genomic pathway necessary for cellular alterations. We herein tested the prediction that specific regulation of ERE-driven genes by an engineered monomeric and constitutively active transcription factor, monotransregulator, provides a basis for the treatment of ER-negative breast cancer. Using adenovirus infected ER-negative MDA-MB-231 cells derived from a breast adenocarcinoma, we found that the monotransregulator, but not the ERE-binding defective counterpart, repressed cellular proliferation and motility, and induced apoptosis through expression of genes that required ERE interactions. Similarly, the monotransregulator suppressed the growth of ER-negative BT-549 cells derived from a breast-ductal carcinoma. Moreover, the ERE-binding monotransregulator repressed xenograft tumor growth in a nude mice model. Thus, specific regulation of genes bearing EREs could offer a therapeutic approach for de novo endocrine-resistant breast cancers.
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Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Receptor alfa de Estrogênio/genética , Elementos de Resposta , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Morte Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reguladores , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Evidence suggests that the CXXC type zinc finger (ZF-CXXC) protein 5 (CXXC5) is a critical regulator/integrator of various signaling pathways that include the estrogen (E2)-estrogen receptor α (ERα). Due to its ZF-CXXC domain, CXXC5 is considered to be a member of the ZF-CXXC family, which binds to unmethylated CpG dinucleotides of DNA and through enzymatic activities for DNA methylation and/or chromatin modifications generates a chromatin state critical for gene expressions. Structural/functional features of CXXC5 remain largely unknown. CXXC5, suggested as transcription and/or epigenetic factor, participates in cellular proliferation, differentiation, and death. To explore the role of CXXC5 in E2-ERα mediated cellular events, we verified by generating a recombinant protein that CXXC5 is indeed an unmethylated CpG binder. We uncovered that CXXC5, although lacks a transcription activation/repression function, participates in E2-driven cellular proliferation by modulating the expression of distinct and mutual genes also regulated by E2. Furthermore, we found that the overexpression of CXXC5, which correlates with mRNA and protein levels of ERα, associates with poor prognosis in ER-positive breast cancer patients. Thus, CXXC5 as an unmethylated CpG binder contributes to E2-mediated gene expressions that result in the regulation of cellular proliferation and may contribute to ER-positive breast cancer progression.
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Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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.
Assuntos
Receptor beta de Estrogênio/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Divisão Celular , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Masculino , Camundongos , Reação em Cadeia da Polimerase , Neoplasias da Próstata/patologia , Interferência de RNA , Receptores de Estrogênio/fisiologia , Transdução de Sinais , Testículo/fisiologiaRESUMO
17beta-estradiol (E2), the main circulating estrogen hormone, is involved in a wide variety of physiological functions ranging from the development to the maintenance of many tissues and organs. The effects of E2 on cells are primarily conveyed by the transcription factors, estrogen receptor (ER) alpha and beta. The regulation of responsive genes by the well-defined ER alpha in response to E2 relies on complex and highly organized processes that dynamically integrate functions of many transcription regulators to induce spatiotemporal alterations in chromatin state and structure. Changes in gene expressions result in cell-specific responses that include proliferation, differentiation and death. Deregulation of E2-ER alpha signaling contributes to the initiation and progression of target tissue malignancies. We aim here to provide a review of recent findings on dynamic transcriptional events mediated by E2-ER alpha with the anticipation that a better understanding of complex regulatory mechanisms underlying ER actions would be a critical basis for the development of effective prognostic tools for and therapeutic interventions against estrogen target tissue malignancies.
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Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Sítios de Ligação/genética , Estradiol/sangue , Receptor alfa de Estrogênio/química , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios ProteicosRESUMO
BACKGROUND: Estrogen receptor alpha (ER alpha) has been reported to be expressed and function in the prostate stromal cells, and numerous evidences indicated that the stromal ER alpha signal pathway plays critical roles in prostate development and cancer. ER alpha requires distinct coregulators for efficient transcriptional regulation. The goal of this study is to examine physical and functional interaction between ER alpha and ERAP75 in the context of prostate stromal cells. METHOD: Yeast two-hybrid assays were used to screen novel ER alpha interaction proteins. The interaction between ER alpha and ERAP75 was confirmed by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation methods. The interaction motif was examined by site-directed mutagenesis. The effect of ERAP75 on ER alpha transactivation and the expression of ER alpha target genes were determined by luciferase assay and real-time PCR, respectively. RESULT: ER alpha can interact with the C terminus of ERAP75 via its ligand binding domain both in vivo and in vitro. The conserved LXXLL motif within the C terminus of ERAP75 is required for the interaction between ER alpha and ERAP75. ERAP75 can enhance ER alpha transactivation in a dose-dependent manner and up-regulate the expression of the endogenous ER alpha target gene, stromal-derived factor-1 (SDF-1), in the prostate stromal cells. CONCLUSION: ERAP75 functions as a novel coactivator that can modulate ER alpha function in the prostate stromal cells. The understanding of the mechanism of ER alpha transactivation in prostate stromal cells could possibly help in the development of new strategies to control or treat prostate cancer by targeting its transactivation protein complex.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Próstata/metabolismo , Células Estromais/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Motivos de Aminoácidos/genética , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/genética , Humanos , Masculino , Pessoa de Meia-Idade , Próstata/citologia , Estrutura Terciária de Proteína/genética , Transdução de Sinais/fisiologia , Células Estromais/citologia , Fatores de Transcrição/genéticaRESUMO
Estrogen hormone (E2) signaling is primarily conveyed by the estrogen receptors (ER) alpha and beta. ERs are encoded by two distinct genes and share varying degrees of domain-specific structural/functional similarities. ERs mediate a complex array of nuclear and non-nuclear events critical for the homeodynamic regulation of various tissue functions. The canonical nuclear signaling involves the interaction of ERalpha and ERbeta with specific DNA sequences, the so-called estrogen responsive elements (EREs). This interaction constitutes the initial step in ERE-dependent signaling in which ERbeta is a weaker transcription factor than ERalpha in response to E2. However, it remains unclear why transactivation potencies of ER subtypes differ. Studies suggest that the amino-terminus, the least conserved structural region, of ERbeta, but not that of ERalpha, impairs the ability of the receptor to bind to ERE independent of E2. Although the impaired ERbeta-ERE interaction contributes, it is not sufficient to explain the weak transactivation potency of the receptor. It appears that the lack of transactivation ability and of the capability of the amino-terminus of ERbeta, as opposed to that of ERalpha, to functionally interact with the carboxyl-terminal hormone-dependent activation domain is also critical for the receptor-specific activity. Thus, the structurally distinct amino-termini of ERs are important determinants in defining the function of ER-subtypes in the ERE-dependent pathway. This could differentially affect the physiology and pathophysiology of E2 signaling.
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Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Transdução de Sinais , Animais , Núcleo Celular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Humanos , Elementos de RespostaRESUMO
The effects of estrogens, particularly 17beta-estradiol (E2), are mediated by estrogen receptor alpha (ERalpha) and ERbeta. 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 ERalpha and ERbeta 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 ERalpha and/or ERbeta 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 ERalpha dimer. These results suggest that ERalpha is the functionally dominant partner in the ERalpha/beta heterodimer.
Assuntos
Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , DNA/metabolismo , Dimerização , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Humanos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta , Ativação TranscricionalRESUMO
The functions of 17beta-estradiol (E2) are mediated by estrogen receptor (ER) alpha and beta. ERs display similar DNA- and ligand-binding properties in vitro. However, ERbeta shows lower transcriptional activity than ERalpha from the estrogen response element (ERE)-dependent signaling. We predicted that distinct amino termini contribute to differences in transcription efficacies of ERs by affecting in situ ER-ERE interactions. We used chromatin immunoprecipitation and a novel in situ ERE competition assay, which is based on the ability of ER to compete for ERE binding with a designer activator that constitutively induces transcription from an ERE-driven reporter construct. Interference of activator-mediated transcription by unliganded or liganded ERs was taken as an indication of ER-ERE interaction. Results revealed that ERs interacted with ERE similarly in the absence of E2. However, E2 enhanced the ERE binding of ERalpha but not that of ERbeta. The removal of the amino terminus increased the ERbeta-ERE interaction independent of E2. The ERbeta amino terminus also prevented E2-mediated enhancement of the chimeric ERalpha-ERE interaction. Thus, the amino terminus of ERbeta impairs the binding of ERbeta to ERE. The abrogation of ligand-dependent activation function 2 of the amino-terminally truncated ERbeta resulted in the manifestation of E2 effect on ERbeta-ERE interaction. This implies that E2-mediated enhancement of ERbeta-ERE interaction is masked by the activation function 2, whereas the intact amino terminus is a dominant region that decreases the binding of ERbeta to ERE. Thus, ERbeta-ERE interaction is independent of E2 and is impaired by its amino terminus. These findings provide an additional explanation for differences between ERalpha and ERbeta functions that could differentially affect the physiology and pathophysiology of E2 signaling.
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Estradiol/metabolismo , Receptor beta de Estrogênio/metabolismo , Elementos de Resposta , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Imunoprecipitação da Cromatina , Cricetinae , Cricetulus , Dimerização , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/química , Receptor beta de Estrogênio/genética , Células HeLa , Humanos , Estrutura Terciária de Proteína/genética , Deleção de Sequência , Ativação TranscricionalRESUMO
17ß-estradiol (E2), the primary circulating estrogen hormone, mediates physiological and pathophysiological functions of breast tissue mainly through estrogen receptor α (ERα). Upon binding to E2, ERα modulates the expression of target genes involved in the regulation of cellular proliferation primarily through interactions with specific DNA sequences, estrogen response elements (EREs). Our previous microarray results suggested that E2-ERα modulates CXXC5 expression. Because of the presence of a zinc-finger CXXC domain (ZF-CXXC), CXXC5 is considered to be a member of the ZF-CXXC family, which binds to non-methylated CpG dinucleotides. Although studies are limited, CXXC5 appears to participate as a transcription factor, co-regulator and/or epigenetic factor in the regulation of cellular events induced by various signaling pathways. However, how signaling pathways mediate the expression of CXXC5 is yet unclear. Due to the importance of E2-ERα signaling in breast tissue, changes in the CXXC5 transcription/synthesis could participate in E2-mediated cellular events as well. To address these issues, we initially examined the mechanism whereby E2-ERα regulates CXXC5 expression. We show here that CXXC5 is an E2-ERα responsive gene regulated by the interaction of E2-ERα with an ERE present at a region upstream of the initial translation codon of the gene.
Assuntos
Proteínas de Transporte/genética , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Transdução de Sinais , Fatores de TranscriçãoRESUMO
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.
Assuntos
Neoplasias da Mama/terapia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Terapia Genética , Humanos , LigantesRESUMO
Estrogen receptors (ER) alpha and beta are members of a superfamily of nuclear receptors and mediate estrogen [17beta-estradiol (E2)] signaling. ERbeta has considerably less transcription potency than ERalpha in heterologous expression systems that use E2 response elements (ERE) in tandem as the trans-acting unit. We show here that despite similar intracellular characteristics, ERbeta, in contrast to ERalpha, fails to induce gene transcription synergistically in response to E2 from tandem EREs. Moreover, our results indicate that ERalpha-specific partial agonistic activity of antagonists occurs additively. Although synergy contributes, it is not sufficient for differences in the transcription potencies between the ER subtypes. We demonstrate here that differences in the abilities of ERs to integrate activation functions through functional interactions between amino and carboxyl termini are critical for the transcriptional strength of ER subtypes.
Assuntos
Receptores de Estrogênio/metabolismo , Animais , Sequência de Bases , Células COS , Linhagem Celular , DNA/genética , DNA/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Genes Reporter , Humanos , Ligantes , Regiões Promotoras Genéticas , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
Estrogen signaling is mediated by ER alpha and -beta. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ER alpha and ER beta bind to an ERE independently from ER ligands. We found that although the binding affinity of ER beta for an ERE is 2-fold lower than that of ER alpha, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ER alpha preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ER alpha is more transcriptionally active than ER beta. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ER alpha to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.
Assuntos
Receptores de Estrogênio/fisiologia , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Estrogênios/fisiologia , Células HeLa , Humanos , Ligantes , Masculino , Dados de Sequência Molecular , Conformação Proteica , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Transdução de Sinais , Ativação TranscricionalRESUMO
Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17ß-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an 'activator' or a 'repressor' possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue.