Corso-Díaz Ximena, de Leeuw Charles N, Alonso Vivian, Melchers Diana, Wong Bibiana K Y, Houtman René, Simpson Elizabeth M
Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, V5Z 4H4, Canada.
Genetics Graduate Program, University of British Columbia, Vancouver, BC, V6T 1Z2, Canada.
BMC Genomics. 2016 Oct 26;17(1):832. doi: 10.1186/s12864-016-3173-5.
NR2E1 (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).
We 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.
We 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.
NR2E1（Tlx）是一种孤儿核受体，可调节神经干细胞的维持和自我更新，并促进肿瘤发生。Nr2e1基因敲除小鼠表现出皮质和边缘结构减少以及明显的视网膜营养不良。NR2E1主要作为基因转录的抑制因子，与共抑制因子萎缩素-1、LSD1、HDAC和BCL11A结合发挥作用。最近的证据表明，NR2E1也可作为基因转录的激活因子。然而，尚未鉴定出与NR2E1相互作用的共激活因子复合物。为了寻找NR2E1潜在的新型共调节因子，我们使用了一种微阵列分析方法，即共调节因子-核受体相互作用实时分析（MARCoNI），该方法包含代表潜在共调节蛋白相互作用基序的肽段，包括已知的共激活因子核受体盒序列（LxxLL基序）。
我们发现NR2E1与用作阳性对照的萎缩素-1肽段（Atro盒）以及构成NR2E1候选伴侣的其他19种肽段有强烈结合。其中两种蛋白，p300和雄激素受体（AR），通过相互免疫沉淀实验进一步得到验证。使用两个单氨基酸变体R274G和R276Q评估了NR2E1与阵列中肽段结合的特异性，这两个变体破坏了野生型NR2E1观察到的大部分结合相互作用。通过以萎缩素-1为诱饵的免疫沉淀实验进一步验证了这些变体与共调节因子结合亲和力的降低。尽管精氨酸274在脊椎动物中高度保守，但通过对携带变体R274G的单拷贝Nr2e1基因敲除小鼠的视网膜表型分析确定，其在体内与共调节因子相互作用的减少并不显著。
我们表明MARCoNI是一种测试NR2E1与候选共调节因子相互作用的特异性分析方法。通过这种方法，我们揭示了19种NR2E1潜在的共调节因子伴侣，包括8种共激活因子。这里鉴定出的所有候选因子都需要使用体外和体内模型进一步验证。该分析方法对NR2E1配体结合域的点突变敏感，有助于识别改变NR2E1与蛋白伴侣结合的突变和/或小分子。
