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

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

Published on: November 15, 2013

Co-activator candidate interactions for orphan nuclear receptor NR2E1.

Ximena Corso-Díaz^1,2, Charles N de Leeuw^1,3, Vivian Alonso¹

¹Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, V5Z 4H4, Canada.

|October 27, 2016

Summary

This study identified 19 novel co-regulator partners for NR2E1 (Tlx), including eight co-activators, using the MARCoNI assay. This reveals new insights into NR2E1

Area of Science:

Molecular Biology
Neuroscience
Genetics

Background:

NR2E1 (Tlx) is an orphan nuclear receptor crucial for neural stem cell maintenance and tumor promotion.
NR2E1 primarily acts as a gene transcription repressor but can also function as an activator.
Identifying NR2E1 co-activators is essential for understanding its diverse regulatory roles.

Purpose of the Study:

To identify novel co-regulators that interact with NR2E1.
To characterize the interaction specificity of NR2E1 with potential partners.
To validate the utility of the MARCoNI assay for NR2E1 co-regulator discovery.

Main Methods:

Utilized microarray assay for real-time analysis of co-regulator-nuclear receptor interaction (MARCoNI).
Screened peptides representing interaction motifs from potential co-regulatory proteins.
Employed reciprocal pull-down assays and site-directed mutagenesis for validation.

Main Results:

Identified 19 potential NR2E1 co-regulator partners, including eight co-activators.
Confirmed interactions with p300 and androgen receptor (AR) via pull-down assays.
Demonstrated assay specificity using NR2E1 variants (R274G, R276Q) that disrupted binding.

Conclusions:

MARCoNI is a specific and sensitive assay for identifying NR2E1 co-regulator interactions.
Discovered novel potential co-activators for NR2E1, expanding the understanding of its function.
The assay can be used to identify mutations or small molecules affecting NR2E1-protein interactions.

Keywords:

Co-regulator MARCoNI NR2E1 Orphan nuclear receptor Peptide array

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