位点特异DNA甲基化调控细胞周期素D1基因

The Transcription Regulation of Site-Specific DNA Methylation on Cyclin D1 Expression

DNA甲基化是基因表达调控的重要方式,一般被认为以CpG岛形式发挥作用;近年的研究表明,位点特异的DNA甲基化以元件(cis-regulatory element)的形式在生物微进化以及基因精细调控中发挥重要作用。我们的研究结果显示,曲古抑菌素A(trichostatin A,TSA)导致细胞周期蛋白D1基因表达下调,以及其核心启动子的+65到+77之间的两段串联CpG序列发生DNA甲基化。报告基因结果显示,由细胞周期素D1核心启动子片段驱动的报告基因活性在TSA处理后下降到原来0.12,而前段(A)突变与后段(B)突变时,报告基因活性分别下降到原来1/7.6与1/8.36,双突变则下降到原来1/7.51。对于CMV启动子嵌合15 bp片段驱动的报告基因活性,野生型、A、B与AB双突变型报告基因活性分别下降1/1.48、1/1.17、1/1.51以及1/1.21。体外甲基化研究结果显示,与对照组相比,M.SssⅠ处理组的启动子活性中,野生型活性下降到原来1/8800,A突变型活性下降到原来1/2700,B突变型活性下降到原来1/4156,双突变型活性下降到原来1/6222。应用M.HhaⅠ处理进一步区分甲基化位点,A和B突变型报告基因活性分别下降到原来1/1.11和1/1.40。有趣的是,比对发现劳亚兽总目的+66位点为T。由此,我们联合应用DNA定点突变与体外DNA甲基化模拟技术,研究了细胞周期蛋白D1基因位点DNA甲基化元件的作用。我们的研究表明,细胞周期蛋白D1基因核心启动子的+65位点的甲基化在转录调控过程中可能发挥关键作用,为位点特异甲基化在基因精细调控的机制研究提供实验数据。

DNA methylation plays an important role on gene expression and is usually thought to function in the CpG islands.Recent studies have shown that site-specific DNA methylation could play important role in transcriptional regulation and biological micro-evolution by the form of cis-regulatory elements.Our study here showed CCND 1 mRNA level decreased and DNA were methylated between+65 and+77 sequence of its core promoter which has two tandem repeats of CpGs under TSA treatment.The activity of reporter gene driven by the DNA fragment of CCND 1 core promoter(wild type,A mutant,B mutant and AB double mutant)reduced by 1.48,1.17,1.51 and 1.21 folds respectively under TSA treatment.DNA methylation modification by M.SssⅠmethyltransferase in vitro caused the decrease of reporter gene activity by 8800,2700,4156 and 6222 folds respectively in case of the DNA fragment of wild type,A,B and AB double mutant.After treatment by M.HhaⅠmethyltransferase to differentiate CpG sites in A and B unit,mutant in A or B unit cause reporter activity decrease 1.11 and 1.40 folds respectively.Interestingly,the single base substitution was found to occur in+66 site of T in laurasiatherian where G is in human.Here in our study,the role of DNA methylation elements in the cyclin D1 locus was studied by combining DNA site-directed mutagenesis with in vitro DNA methylation simulation techniques.The results showed that the+65 methylation site may play the key role in CCND 1 transcription.Our data would provide a clue for site-specific methylation in the mechanism of gene regulation.