ANALYSIS OF CYCLIN D1 GENE EXPRESSION BY METHYLATION OF CpG ISLAND LOCATED 5'-FLANKING REGION

我们用分子病理学技术精确定位了大鼠细胞周期蛋白D1基因5 ′侧翼区甲基化胞嘧啶。在我们实验室建立的一系列化学致癌剂诱导的大鼠白血病细胞系中，我们发现细胞周期蛋白D1整合素的表达在测试的细胞系中存在差异。我们在这些细胞系中没有发现基因扩增和重排。因此，我们克隆了含有cyclin D1基因启动子的5 '侧翼区，以阐明过表达的机制。当我们对老鼠的基因组DNA进行测序时，我们发现cyclinD1基因启动子含有两个连续的Sp1位点，并含有大量的CpG位点。因此我们推测CpG甲基化可能在调控cyclin D1基因稳态表达中起重要作用。首先我们通过限制性内切酶消化和Southern印迹分析来筛选甲基化状态。正如我们所预期的，我们发现，严重的CpG甲基化导致细胞周期蛋白D1基因表达的抑制在稳态水平。此外，CpG位点的去甲基化，特别是在两个Spl位点附近的去甲基化上调了cyclin Dl基因的稳态表达。我们的数据在各种国际会议以及国内会议上发表，并在学术期刊上发表了大量的科学论文。

We have shown precise localization of methylated cytosine in the 5'-flanking region of rat cyclin D1 gene by molecular pathological technique. In a series of chemical carcinogen-induced rat leukemic cell lines established in our laboratory, we found that the expression of the cyclin D1 integrin varied among cell lines tested. We found no gene amplification and rearrangement in those cell lines. We therefore cloned 5'-flanking region containing cyclin Dl gene promoter to elucidate the mechanism of overexpression. When we sequenced rat genomic DNA,. we found that cyclin Dl gene promoter contained two continuous Spl sites with numerous CpG loci. We therefore speculated that CpG methylation might play an important role in the regulation of the cyclin D1 gene expression at steady-state level, First we screened the methylation status by restriction enzymatic digestion followed by Southern blot analysis. As we expected, we found that heavy CpG methylation resulted in the suppression of the cyclin D1 gene expression at the steady-state level. Furthermore, demethylation of the CpG loci especially around the two Spl sites up-regulated the steady-state expression of cyclin Dl gene.We have presented our data at various international congress as well as domestic meetings, and publish a lot of scientific papers for academic journals.