The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites
Part of the
Methods in Molecular Biology
book series (MIMB, volume 1366)
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.
Several 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.
Key wordsEstrogenreceptoralpha (ERα) Estrogen transcriptional regulation Estrogen-responsive element (ERE) Nonclassical pathway AP-1pathway Estrogenreceptor alanine 87 polymorphism
This work was partially supported by Fondo Clemente Estable, ANII (Uruguay), and PEDECIBA.
Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240(4854):889–895CrossRefGoogle Scholar
Merot Y, Metivier R, Penot G et al (2004) The relative contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha transcriptional activity depends upon the differentiation stage of the cell. J Biol Chem 279(25):26184–26191CrossRefGoogle Scholar
Nilsson S, Makela S, Treuter E et al (2001) Mechanisms of estrogen action. Physiol Rev 81(4):1535–1565CrossRefGoogle Scholar
Welboren WJ, Sweep FC, Span PN, Stunnenberg HG (2009) Genomic actions of estrogen receptor alpha: what are the targets and how are they regulated? Endocr Relat Cancer 16(4):1073–1089CrossRefGoogle Scholar
Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL (2001) Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway. J Biol Chem 276(17):13615–13621CrossRefGoogle Scholar
Gennari L, Merlotti D, De Paola V et al (2005) Estrogen receptor gene polymorphisms and the genetics of osteoporosis: a HuGE review. Am J Epidemiol 161(4):307–320CrossRefGoogle Scholar
Mill J, Kiss E, Baji I et al (2008) Association study of the estrogen receptor alpha gene (ESR1) and childhood-onset mood disorders. Am J Med Genet B Neuropsychiatr Genet 147B(7):1323–1326CrossRefGoogle Scholar
Lewis JS, Jordan VC (2005) Selective estrogen receptor modulators (SERMs): mechanisms of anticarcinogenesis and drug resistance. Mutat Res 591(1-2):247–263CrossRefGoogle Scholar
Berry M, Metzger D, Chambon P (1990) Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9(9):2811–2818CrossRefGoogle Scholar
Fan JD, Wagner BL, McDonnell DP (1996) Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity. Mol Endocrinol 10(12):1605–1616PubMedGoogle Scholar
Safe S, Kim K (2008) Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 41(5):263–275CrossRefGoogle Scholar
Kushner PJ, Agard DA, Greene GL et al (2000) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74(5):311–317CrossRefGoogle Scholar
Fernandez-Calero T, Astrada S, Alberti A et al (2014) The transcriptional activities and cellular localization of the human estrogen receptor alpha are affected by the synonymous Ala87 mutation. J Steroid Biochem Mol Biol 143C:99–104CrossRefGoogle Scholar
© Springer Science+Business Media New York 2016