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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

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

B A Laffitte1, H R Kast, C M Nguyen

  • 1Departments of Biological Chemistry, UCLA, Los Angeles, California 90095, USA.

The Journal of Biological Chemistry
|April 1, 2000
PubMed

Insights

Researchers identified the optimal DNA binding sequence for the farnesoid X-activated receptor (FXR) and 9-cis-retinoic acid receptor (RXR) heterodimer. This consensus sequence, an inverted repeat (IR-1), facilitates FXR/RXR binding and gene activation, aiding in the discovery of new target genes.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The farnesoid X-activated receptor (FXR) is a nuclear hormone receptor that forms a heterodimer with the 9-cis-retinoic acid receptor (RXR).
  • Understanding the DNA binding preferences of the FXR/RXR heterodimer is crucial for elucidating gene regulation by FXR.

Purpose of the Study:

  • To determine the optimal DNA binding sequence for the FXR/RXR heterodimer.
  • To identify functional response elements and target genes regulated by FXR/RXR.

Main Methods:

  • Selected and amplified binding sequence imprinting technique to identify DNA binding sequences.
  • Electrophoretic mobility shift assays (EMSA) to assess DNA binding affinity.
  • Transient transfection assays to evaluate ligand-dependent transcriptional activation.

Main Results:

  • The consensus optimal DNA binding sequence for FXR/RXR is an inverted repeat with a 1-base pair spacing (IR-1).
  • FXR/RXR binds with high affinity to the IR-1 sequence and can also bind to related sequences with variations.
  • Three genes containing IR-1 elements in their promoters were identified as FXR/RXR targets, with phospholipid transfer protein mRNA levels shown to be induced by FXR ligands.

Conclusions:

  • The IR-1 sequence and its variants are high-affinity binding sites and functional response elements for the FXR/RXR heterodimer.
  • Identification of phospholipid transfer protein as a target gene provides insight into FXR-mediated gene regulation.
  • These findings will facilitate the discovery of additional genes regulated by FXR/RXR.

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