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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
10:51

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

Published on: November 15, 2013

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

Masae Ohno1, Masaaki Kunimoto, Makoto Nishizuka

  • 1Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan.

Biochemical and Biophysical Research Communications
|October 17, 2009
PubMed

Insights

The Ku proteins (Ku70 and Ku80) interact with the farnesoid X receptor (FXR) and act as corepressors, inhibiting bile acid metabolism gene expression. This discovery reveals a novel regulatory mechanism for FXR.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The farnesoid X receptor (FXR) is a nuclear receptor crucial for regulating bile acid metabolism and enterohepatic circulation.
  • FXR's function is modulated by various cofactors, but the specific roles of DNA repair proteins in FXR regulation remain largely unexplored.

Purpose of the Study:

  • To identify novel cofactors interacting with FXR.
  • To elucidate the functional role of identified cofactors in FXR-mediated gene regulation, particularly concerning bile acid metabolism.

Main Methods:

  • Pull-down assays using GST-fused FXR regions (A/B and C) with HeLa cell nuclear extracts.
  • Identification of interacting proteins via pull-down assays.
  • Chromatin immunoprecipitation (ChIP) assays to assess protein-DNA interactions on the Bile Salt Export Pump (BSEP) promoter.
  • Reporter gene assays to evaluate the effect of Ku proteins on FXR-mediated BSEP promoter activity.

Main Results:

  • DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70, and Ku80 were identified as FXR-associated factors.
  • Ku70 and Ku80 interacted with the C and D regions of FXR, while DNA-PKcs interacted with the A/B region.
  • Ku proteins were found to associate with the BSEP promoter in conjunction with FXR.
  • Ectopic expression of Ku proteins suppressed FXR-mediated BSEP promoter activity and gene expression.

Conclusions:

  • Ku70 and Ku80 function as corepressors for FXR.
  • These findings reveal a novel role for DNA repair proteins, Ku70 and Ku80, in the regulation of bile acid metabolism via FXR signaling.

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