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

2.转录抑制因子N-COR的结构域分析我们产生了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及其活性的抑制。3.Runx2雄激素受体的相互反式激活抑制它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都通过从另一种蛋白的原始区段中提取它来抑制另一种蛋白的转录功能。4.构建高发光YFP-cDNA库并将其导入培养细胞。从HeLa细胞中提取信使RNA，用逆转录酶合成cDNA，将其与高发光YFP载体融合，形成融合文库。将该文库与AR表达载体共转染COS-7细胞。在配体治疗后，我们试图收集显示出与AR相似的特定核模式的细胞。然而，我们未能拾取这种细胞，因为呈现灶状图案的细胞非常罕见。

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.