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
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.