title: The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.
abstract: Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.
authors.affiliation: Biochemistry-Molecular Biology, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400, Montevideo, Uruguay. tamfer@pasteur.edu.uy.|Bioinformatics Unit, Institut Pasteur Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay. tamfer@pasteur.edu.uy.
authors.forename: Tamara
authors.initials: T
authors.lastname: Fernández-Calero
authors.affiliation: Université de Rennes 1, Institut de Recherche en Santé, Environnement et Travail, IRSET, INSERM U1085, Team TREC, Biosit, Rennes, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Flouriot
authors.affiliation: Biochemistry-Molecular Biology, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400, Montevideo, Uruguay.
authors.forename: Mónica
authors.initials: M
authors.lastname: Marín
mesh_terms: D000409:Alanine; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D020218:Response Elements; D013937:Thymidine Kinase / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D057188:Workflow
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D018808:Transcription Factor AP-1; D008156:Luciferases; D013937:Thymidine Kinase; D000409:Alanine
keywords: AP-1 pathway; Estrogen -responsive element (ERE ); Estrogen receptor alanine 87 polymorphism; Estrogen receptor alpha (ERα ); Estrogen transcriptional regulation; Nonclassical pathway