Comprehensive identification of nuclear receptor-regulating proteins usinghigh luminescent YFP-fusion cDNA library

1. 甾体激素受体核病灶形成的意义研究我们发现，甾体激素受体的配体依赖性病灶形成并不先于转录激活，而是取决于共激活因子和共抑制因子的相对表达比例。转录压缩器N-CoR结构域分析我们生成了多种N-CoR缺失突变体，发现该蛋白的中心部分（1133-1798氨基酸）主要负责与类固醇激素受体的相互作用并抑制其活性。为了研究类固醇激素受体在骨代谢中的作用，我们分析了类固醇激素受体与Runx2之间的相互作用，Runx2是成骨细胞分化和增殖的关键调节因子。我们的实验结果表明，AR和Runx2都通过将另一种蛋白从其原始区室中提取出来来抑制其转录功能。高发光YFP-cDNA文库的构建及导入培养细胞从HeLa细胞中提取信使RNA，通过逆转录酶合成cDNA，与高发光YFP载体融合，形成融合文库。将该cDNA文库与ar表达质粒共转染COS-7细胞。在配体处理后，我们试图收集与AR类似的特定核模式的细胞，但由于呈现局灶样模式的细胞非常罕见，我们未能收集到此类细胞。

1. Examination of the significance of nuclear foci formation of steroid hormone receptors We hound that ligand-dependent foci formation of steroid hormone receptors does not precede the transcriptional activation and depends on the relative expression ratio of coactivators and corepressors.2.Domain analysis of transcription compressor N-CoR We generated various N-CoR deletion mutants and hound that the central portion (1133-1798 amino acid) of the protein was mostly responsible for both the interaction with steroid hormone receptors and the repression of their activities.3.Mutual transactivational repression of Runx2 the androgen receptor (AR) Tb examine the role of steroid hormone receptors on bone metabolism, we analized mutual action between steroid hormone receptors and Runx2, which is a key regulator for osteoblast diffrentiation and proliferation. Our experimental results suggest that both AR and Runx2 repress the transcriptional function of the other protein by extracting it from its original compartment.4.Construction of high luminescentYFP-cDNAlibrary and introdution into culture cells Messenger RNA was extracted from HeLa cells and cDNA was synthesized by reverse transcriptase and then fused to high luminescent YFP vector to make fusion library. This cDNA library was cotransfected with AR-expression plasmid into COS-7 cells. After the ligand treatment, we tried to collect cells showing specific nuclear patterns similar to those of AR. However, we failed to pick up such kind of cells because cells showing foci-like pattern was very rare.