Identification of the DNA binding specificity and potential target genes for the farnesoid X-activated receptor.
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 amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR/RXR in vitro. The consensus 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 binding site for FXR/RXR in vitro and to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitro and to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response 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 elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.
Laffitte
B A
BA
Departments of Biological Chemistry, UCLA, Los Angeles, California 90095, USA.
Kast
H R
HR
Nguyen
C M
CM
Zavacki
A M
AM
Moore
D D
DD
Edwards
P A
PA
D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D016415:Sequence Alignment; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099150:Receptor, Farnesoid X-Activated
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated