title: A distal estrogen responsive element upstream the cap site of human transthyretin gene is an enhancer-like element upon ERα and/or ERβ transactivation.
abstract: Previous studies reported that 17 beta-estradiol (E2) is responsible for the up-regulation of transthyretin (TTR) expression via an estrogen receptor (ER)-dependent pathway in rat choroid plexus (CP) and liver. A computer-assisted homology search identified a putative estrogen-responsive element (ERE) in the 5' flanking region of the human TTR (hTTR) gene (ERETTR), with the sequence aAGTCAAAGTGACCa, between -3406 and -3392 bp. Luciferase reporter assays and electrophoretic mobility shift (EMSA) and supershift analysis were carried out to investigate if E2 regulates TTR transcription via this putative ERE. Luciferase reporter assays in COS-7 cells were carried out with a plasmid construction where the TTR fragment containing the putative ERETTR was cloned in pGL2-promoter vector (pGL2-P) (pGL2-P/TTR), co-transfected with estrogen receptor α (ERα) and/or estrogen receptor β (ERβ) expression vectors. These assays demonstrated that, upon incubation with E2, one or both ERs (α and/or β) transactivate the reporter gene. The pGL2-P/TTR showed a significant transactivation of up to 6.8-fold, by E2, when co-transfected with ERβ, and up to 4-fold with ERα. Specific binding of ER (α and/or β) to ERETTR was demonstrated by EMSA and supershift assays confirmed the binding to ERα and/or ERβ. Our findings further suggest a mechanism underlying the regulation of TTR expression through the identification of a novel ERE in the TTR gene, which functions as an E2-dependent enhancer-like element.
authors.affiliation: CICS - UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, Covilhã 6200-506, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Martinho
authors.forename: C R
authors.initials: CR
authors.lastname: Santos
authors.forename: I
authors.initials: I
authors.lastname: Gonçalves
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D047628:Estrogen Receptor alpha / Q000235:genetics*; D047629:Estrogen Receptor beta / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D011228:Prealbumin / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D011228:Prealbumin
keywords: 17 beta-estradiol; B-gal; CP; CSF; E2; EMSA; ER; ERE; ERETTR cloned in pGL2 vector; EREcon; ERα; ERβ; Enhancer; Estradiol; Estrogen receptors; Estrogen responsive element; RBP; TTR; Transthyretin; beta-galactosidase; cerebrospinal fluid; choroid plexus; consensus oligonucleotide for ERE; electrophoretic mobility shift assay; estrogen receptor; estrogen receptor alpha; estrogen receptor beta; estrogen-responsive element; pGL2-P; pGL2-P/TTR; pGL2-promoter vector; retinol binding protein; transthyretin