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Analysis of DNA methylation pattern of promoter CpG islands in idiopathic pulmonary fibrosis.

特发性肺纤维化启动子CpG岛DNA甲基化模式分析

基本信息

批准号：
15390257
负责人：
HOMMA Yoshimi
金额：
$ 9.54万
依托单位：
FUKUSHIMA MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Methylation of cytosine residues of the CpG dinucleotides constitutes the basis of an epigenetic control of gene expression in mammals. Genomic methylation patterns are of critical importance in various biological processes such as development, imprinting, and tumorigenesis. Most cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific-GC rich loci, called CpG islands. These small stretches of DNA sequences are located in the promoter and first exon regions of 60% of human genes, and methylation of cytosine in these CpG islands is associated with loss of gene expression. Although much evidence has been accumulated to show aberrant de novo methylation in cancer, regulation of maintenance and de novo methylation in normal and other diseases is obscure. In this study, we developed a new microarray-based method to detect differences in methylation patterns of the promoter CpG of 360 genes including transcription factors and signaling molecules. Using this microarray, we analyzed genome samples derived from patients with idiopathic pulmonary fibrosis (IPF). Fluorescent probes were prepared from genome samples obtained from patients and normal donors by a methylation amplification method. After hybridization, array data was analyzed to compare the methylation states of two groups. Differences were detected at 〜25 loci, suggesting differential methylation states in patients with IPF. We, then, analyzed methylation of each CpG site of these loci by a bisulfite sequencing method, and detected several genes whose promoter CpG is hypomethylated in IPF. These results suggest a possible involvement of epigenetic factors in pathogenesis of IPF, and we examine the protein levels of these genes now.

CpG二核苷酸的胞嘧啶残基的甲基化构成哺乳动物中基因表达的表观遗传控制的基础。基因组甲基化模式在各种生物学过程如发育、印记和肿瘤发生中至关重要。CpG二核苷酸内的大多数胞嘧啶在人类基因组中是甲基化的，但一些在称为CpG岛的富含特异性GC的基因座中保持未甲基化。这些DNA序列的小片段位于60%的人类基因的启动子和第一外显子区域，并且这些CpG岛中胞嘧啶的甲基化与基因表达的丧失相关。尽管已经积累了大量证据表明癌症中异常的从头甲基化，但正常和其他疾病中维持和从头甲基化的调节尚不清楚。在这项研究中，我们开发了一种新的基于微阵列的方法来检测360个基因，包括转录因子和信号分子的启动子CpG甲基化模式的差异。使用这种微阵列，我们分析了来自特发性肺纤维化（IPF）患者的基因组样本。通过甲基化扩增方法从患者和正常供体获得的基因组样品制备荧光探针。杂交后，分析阵列数据以比较两组的甲基化状态。在25个位点检测到差异，表明IPF患者的甲基化状态不同。然后，我们通过亚硫酸氢盐测序法分析了这些位点的每个CpG位点的甲基化，并检测了IPF中启动子CpG低甲基化的几个基因。这些结果表明表观遗传因素可能参与了特发性肺纤维化的发病机制，我们现在检查了这些基因的蛋白水平。