Abstract
BackgroundNR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis. Nr2e1-null mice exhibit reduced cortical and limbic structures and pronounced retinal dystrophy. NR2E1 functions mainly as a repressor of gene transcription in association with the co-repressors atrophin-1, LSD1, HDAC and BCL11A. Recent evidence suggests that NR2E1 also acts as an activator of gene transcription. However, co-activator complexes that interact with NR2E1 have not yet been identified. In order to find potential novel co-regulators for NR2E1, we used a microarray assay for real-time analysis of co-regulator–nuclear receptor interaction (MARCoNI) that contains peptides representing interaction motifs from potential co-regulatory proteins, including known co-activator nuclear receptor box sequences (LxxLL motif).ResultsWe found that NR2E1 binds strongly to an atrophin-1 peptide (Atro box) used as positive control and to 19 other peptides that constitute candidate NR2E1 partners. Two of these proteins, p300 and androgen receptor (AR), were further validated by reciprocal pull-down assays. The specificity of NR2E1 binding to peptides in the array was evaluated using two single amino acid variants, R274G and R276Q, which disrupted the majority of the binding interactions observed with wild-type NR2E1. The decreased binding affinity of these variants to co-regulators was further validated by pull-down assays using atrophin1 as bait. Despite the high conservation of arginine 274 in vertebrates, its reduced interactions with co-regulators were not significant in vivo as determined by retinal phenotype analysis in single-copy Nr2e1-null mice carrying the variant R274G.ConclusionsWe showed that MARCoNI is a specific assay to test interactions of NR2E1 with candidate co-regulators. In this way, we unveiled 19 potential co-regulator partners for NR2E1, including eight co-activators. All the candidates here identified need to be further validated using in vitro and in vivo models. This assay was sensitive to point mutations in NR2E1 ligand binding domain making it useful to identify mutations and/or small molecules that alter binding of NR2E1 to protein partners.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3173-5) contains supplementary material, which is available to authorized users.
Highlights
NR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis
We found that FLAG-NR2E1LBD interacted very strongly with the Atro-box peptide in the array but bound very poorly to the mutant Atro box (Fig. 1; p < 0.05, n = 3 arrays)
We further studied the ability of FLAG-NR2E1LBD R274G or R276Q to interact with human atrophin-1 by affinity purification assays
Summary
NR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis. Nuclear receptors interact with multi-protein coactivator or co-repressor complexes to activate or repress transcription, respectively. These co-regulator complexes have histone modification and chromatin remodeling functions that elicit transcriptional control. NR2E1 can act as an activator of gene transcription by binding to the promoters of Wnt7a [1], and Mash1 [17]. Despite all this knowledge, the composition and dynamics of the co-repressor complexes formed by NR2E1 are not well understood and, to date, no co-activator proteins have been found to interact with NR2E1. The crystal structure of NR2E1 in complex with a short sequence from atrophins (Atro box) was recently resolved showing that the autorepressed conformation of NR2E1 creates a pocket to recruit atrophins, which bind to NR2E1 through an unconventional sequence: ALxxLxxY [18]
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