MOLECULAR MECHANISMS OF METALLOTHIONEIN INDUCTION

描述(改编自申请者的摘要)：本实验室有 证明过表达大亚基的大鼠成纤维细胞 蛋白Ku(Ku-80)抑制重毒诱导的金属硫蛋白(MT) 金属，而亲本细胞(大鼠1)或过度表达小分子 两个亚基的Ku亚基(Ku-70)作为杂二聚体(Ku-7080)与 诱导MT。进一步的研究表明，这种压抑是由 MT启动子的高甲基化，可能是通过诱导一种可扩散的 可以独立于染色质发挥作用的抑制物(R)。现在 提案旨在阐明负责以下工作的机制 MT-1启动子高甲基化及其与甲基C的作用机制 结合蛋白(MeCP)和受体介导mt-1基因表达的下调。 亚硫酸氢盐基因组测序显示，与大鼠1号细胞不同，所有21个CpG 在Ku-80细胞中，MT-1立即启动子中的二核苷酸发生甲基化。这 工作将确定甲基C结合蛋白(MeCP)的性质(激活 或诱导)通过紫外光交联和西南印迹分析 以甲基化DNA为探针，进行蛋白质印迹分析。如果MeCP激活或 在KU-80细胞中诱导的不同于两种特征的MeCP，它将是 纯化和克隆，鉴定了抑制物结构域，并观察了 确定了甲基化MT启动子转录的重组MeCP，以及 它抑制转录的机制将被阐明。接下来， 研究人员将探索信号转导机制， Ku亚基的过表达增强了MT-1启动子的甲基化， 特别是在提高DNA甲基转移酶活性和 H-ras可能在介导该酶活性升高中的作用。R 将是除MeCP之外的特征，并将尝试 阐明其抑制MT-1转录的机制。此外， 与不表达MT且P80水平较高的肿瘤细胞相比 至p70将被调查。众所周知，MT可以保护细胞免受损害 产生氧自由基并引起氧化的有毒金属和试剂 压力。由于MT的基础水平在大多数组织中可以忽略不计，其 戏剧性的诱导(高达50-100倍，取决于诱导者和 细胞类型)似乎对细胞抵抗有害物质的生存至关重要 这些毒剂的作用。希望本文的研究能对设计有所帮助。 信号转导失活增强MT诱导的途径 导致MT启动子高甲基化的反应。因为MT 由于高甲基化，诱导在许多癌细胞中被抑制， MT的重新表达可能是阻止恶性生长的有效手段之一 这些细胞。

DESCRIPTION (Adapted from the applicant's abstract): This laboratory has demonstrated that rat fibroblast cells that overexpress the large subunit of the protein Ku (Ku-80) repress metallothionein (MT) induction by heavy toxic metals, whereas the parental cells (Rat 1) or cells that overexpress the small Ku subunit (Ku-70) of both subunits as a heterodimer (Ku-7080) conintue to induce MT. Further study revealed that this repression is caused by hypermethylation of MT promoter, and probably by the induction of a diffusible repressor (R) that can function independent of the chromatin. The present proposal is aimed at elucidation of the mechanisms responsible for hypermethylation of MT-1 promoter and the mechanisms by with the methyl C binding protein (MeCP) and R mediate the rpression of Mt-1 gene expression. Bisulfite genomic sequencing showed that unlike Rat-1 cells, all 21 CpG dinucleotides in MT-1 immediate promoter are methylated in Ku-80 cells. This work will identify the nature of the methyl C-binding protein (MeCP) (activated or induced) by UV crosslinking and Southwestern blotting analysis using methylated DNA as probe, and Western blot analysis. If the MeCP activated or induced in Ku-80 cells is distinct from the two characterized MeCPs, it will be purified and cloned, the repressor domain identified, the effect of the recombinant MeCP on transcription from methylated MT promoter determined, and the mechanism by which it inhibits transcription will be elucidated. Next, the investigators will explore the signal transduction mechanisms by which overexpression of Ku subunit enhances methylation of MT-1 promoter, particularly with regard to enhancement of DNA methyltransferase activity and the probably role of H ras in mediating the increase in this enzyme activity. R will be characterized in addition to MeCP, and attempts will be made to elucidate the mechanisms by which it represses MT-1 transcription. Furthermore, tumor cells that do not expresss MT and exhibit higher levels of P80 compared to p70 will be investigated. MT is known to protect cells from damage against toxic metals and agents that generate free oxygen radicals and cause oxidative stress. Since the basal level of MT is negliglible in most tissues, its dramatic induction (as much as 50-100 fold, depending upon the inducer and the cell type) appears to be crucial for the survival of cells against the harmful effects of these agents. It is hoped that the present study will help design ways to augment MT induction by the inactivation of the signal transduction reactions that lead to hypermethylation of the MT promoter. Because MT induction is repressed in many cancer cells due to hypermethylation, the re-expresssion of MT may be one effective means to arrest malignant growth of these cells.