Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor

Citation
Ba. Laffitte et al., Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor, J BIOL CHEM, 275(14), 2000, pp. 10638-10647
Citations number
48
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
14
Year of publication
2000
Pages
10638 - 10647
Database
ISI
SICI code
0021-9258(20000407)275:14<10638:IOTDBS>2.0.ZU;2-T
Abstract
The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis- retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selected and am plified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR /RXR in vitro. The con sensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity b inding site for FXR/RXR in vitro and to confer Ligand-dependent transcripti onal activation by FXR/RXR to a heterologous promoter. Electrophoretic mobi lity shift assays and transient transfection assays were used to investigat e the importance of the core half-site sequences, spacing nucleotide, flank ing sequences, and orientation and spacing of the core half-sites on DNA bi nding and ligand-dependent transcriptional activation by FXR/RXR. These stu dies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activa te through a variety of elements including IR-1 elements with changes in th e core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Th ree genes were identified that contain IR-1 sequences in their proximal pro moters. These elements were shown to bind FXR/RXR in vitro and to confer FX R/RXR-dependent transcriptional activation to a heterologous promoter in re sponse to a bile acid or synthetic retinoid, The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related element s as high affinity binding sites and functional response elements for FXR/R XR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.