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

使用高发光 YFP 融合 cDNA 文库全面鉴定核受体调节蛋白

基本信息

批准号：
18591027
负责人：
KAWATE Hisaya
金额：
$ 2.49万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591027/
关键词：
Nuclear receptor Gene Confocal microscope Hormone

项目摘要

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.

1.类固醇激素受体核灶形成的意义研究我们发现类固醇激素受体核灶的形成并不先于转录激活，而是依赖于辅激活子和辅抑制子的相对表达比例。2.转录压缩子N-CoR的结构域分析我们构建了多种N-CoR缺失突变体，发现其中心部分缺失，而转录激活子和辅抑制子的相对表达比例对核灶的形成有重要影响。（1133-1798氨基酸）主要负责与类固醇激素受体的相互作用和抑制其活性。3.雄激素受体（AR）Runx 2的相互反式激活抑制Tb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和Runx 2之间的相互作用，其是成骨细胞分化和增殖的关键调节剂。我们的实验结果表明AR和Runx 2都是通过将另一个蛋白质从其原来的区域中提取出来而抑制其转录功能的。4.高发光YFP-cDNA文库的构建和导入培养细胞从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，制备融合文库。将该cDNA文库与AR表达质粒共转染COS-7细胞。在配体处理后，我们试图收集显示与AR相似的特定核模式的细胞。然而，我们未能挑选出这种细胞，因为表现出病灶样图案的细胞非常罕见。