title: Usingin vivoelectroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3.
abstract: The technique ofin vivoelectroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp - 612 and - 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the - 612 and - 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct byin vivoelectroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cellsin vitrothus validating this technique forin vivostudies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3treatment.
authors.affiliation: Department of Pharmaceutical Sciences, College of Arts and Sciences, Biomanufacturing Research Institute and Technology Enterprise (BRITE), North Carolina Central University, Durham, NC 27707, USA.
authors.forename: Dayami
authors.initials: D
authors.lastname: Lopez
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Brooks
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Lindsey R
authors.initials: LR
authors.lastname: Boone
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Gene C
authors.initials: GC
authors.lastname: Ness
keywords: CPT-I, carnitine palmitoyltransferase-I; ChIP, chromatin immunoprecipitation; DbMt, double mutant; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assays; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; Hx, hypophysectomized; IP, immunoprecipitation; LDL receptor; LDL, low density lipoprotein; LDLR, LDL receptor; NR, normal; NcoR2, nuclear receptor co-repressor 2; PCR, polymerase chain reaction; PPARα, peroxisomal proliferator receptor α; Peroxisomal proliferator receptor α; Pol II, polymerase II; RA, 9-cis retinoic acid; ROI, region of interest; RXRα, retinoic acid X receptor α; Retinoic acid X receptor α; SEM, standard error of the mean; SMRTe, silencing mediator for retinoid and thyroid hormone receptors-extended; SREBP, sterol response element binding protein; T3, l-triiodothyronine; THDS, thyroid hormone depleted serum; TRAC-1, T3 receptor-associating cofactor 1; TRAP, thyroid hormone receptor- associated protein; TREs, thyroid response elements; TRβ1, thyroid receptor β1; Thyroid hormone receptor β1; Thyroid response element; USF-2, upstream factor-2; Upstream factor-2; WT, wild-type; fT3, free T3; in vivo electroporation; l-triiodothyronine; siRNA, silencing RNA; − 156Mt, − 156 mutant; − 612Mt, − 612 mutant