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Analysis of the Regulatory Mechanism of Transcription by Acyl-CoA Oxidase Gene Enhancer

酰基辅酶A氧化酶基因增强子转录调控机制分析

基本信息

批准号：
06454658
负责人：
OSUMI Takashi
金额：
$ 4.74万
依托单位：
Himeji Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454658/
关键词：
Peroxisomes Acyl-CoA oxidase Transcriptional regulation Peroxisome proliferator PPAR RXR Enhancer Nuclear receptor

项目摘要

1. To investigate the mechanism of induction of peroxisomal functions by peroxisome proliferators, we analyzed the enhancer of the gene for rat acyl-CoA oxidase, the key enzyme of the peroxisomal beta-oxidation system. By gene transfection to cultured animal cells from hepatic and non-hepatic origins, we found that both the direct repeat motif (DR-1) in the enhancer region and its downstream sequence are important for the transcriptional activation. These sequences were revealed to be necessary for the binding with the heterodimer of PPAR and RXR,nuclear receptors both involved in the activation of the gene.2. Roles of the DR-1 downstream sequence were extensively analyzed by transfection and protein binding assays using various mutant enhancer sequences. We found that the four nucleotide sequence next to the DR-1 motif is imortant for both the enhancer activity and PPAR/RXR binding. By selection of high-affinity binding sites from a random sequence pool, a consensus sequence for PPAR/RXR binding, A・G・A or T・T was identified. Furthermore, we showed that PPAR binds to the 3', whereas RXR to the 5'half-site of the acyl-CoA oxidase gene DR-1. This means that the extended half-site motif on the 3' side is required for binding of PPAR.The polarity of binding and requirement for an extended half-site are unique to PPAR,not observed for other heterodimer-forming nuclear receptors.3. We cloned three new proteins interacing with PPAR,by yeast two-hybrid system. One of them belongs to the nuclear hormone receptor superfamily, but lacks a Zn-finger DNA-binding domain at the N-terminus. This protein also interacts with other nuclear receptors, and thus this protein might modulates the functions of various nuclear receptors.4. We observed that PPAR and HNF-4 functionally competes with each other in the transcriptional regulation. We also found that the N-terminal domain of PPAR carries an independent gene activation function.

1. 为了研究过氧化物酶体增殖剂诱导过氧化物酶体功能的机制，我们分析了过氧化物酶体β-氧化系统的关键酶大鼠酰基辅酶A氧化酶基因的增强子。通过对肝源和非肝源的培养动物细胞进行基因转染，我们发现增强子区的同向重复基序（DR-1）及其下游序列对于转录激活很重要。这些序列被揭示是与PPAR和RXR异二聚体结合所必需的，核受体均参与该基因的激活。2．使用各种突变增强子序列通过转染和蛋白质结合测定对 DR-1 下游序列的作用进行了广泛分析。我们发现 DR-1 基序旁边的四个核苷酸序列对于增强子活性和 PPAR/RXR 结合都很重要。通过从随机序列池中选择高亲和力结合位点，确定了 PPAR/RXR 结合的共有序列 A·G·A 或 T·T。此外，我们发现 PPAR 与酰基辅酶 A 氧化酶基因 DR-1 的 3' 半位点结合，而 RXR 与酰基辅酶 A 氧化酶基因 DR-1 的 5' 半位点结合。这意味着PPAR的结合需要3'端延伸的半位点基序。结合的极性和对延伸半位点的要求是PPAR所独有的，在其他形成异二聚体的核受体中没有观察到。3．我们通过酵母二杂交系统克隆了三种与PPAR相互作用的新蛋白。其中之一属于核激素受体超家族，但在 N 末端缺乏锌指 DNA 结合结构域。该蛋白还与其他核受体相互作用，因此该蛋白可能调节多种核受体的功能。4．我们观察到 PPAR 和 HNF-4 在转录调控方面相互竞争。我们还发现PPAR的N端结构域具有独立的基因激活功能。