Genome Wide Analysis of DNA Methylation

DNA 甲基化的全基因组分析

• 批准号: 6878427
• 负责人: Joseph R Ecker
• 金额: $ 50万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-29 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6878427
• 关键词: Arabidopsis; DNA methylation; affinity chromatography; antisense nucleic acid; bioinformatics; biotechnology; functional /structural genomics; gene induction /repression; gene mutation; genetic library; high throughput technology; immunoprecipitation; method development; microarray technology; nucleic acid purification; nucleic acid sequence; oligonucleotides; restriction endonucleases; sulfites; technology /technique development; transposon /insertion element

DESCRIPTION (provided by applicant): This proposal aims to identify functional, non-coding elements in eukaryotic genomes by surveying genome-wide DNA methylation. Cytosine DNA methylation controls developmental gene expression in mammals during genomic imprinting and X-chromosome inactivation, and also silences transposons and other repeated DNA sequences. Hypermethylation of specific tumor suppressor genes contributes to cancer, showing the relevance of this work to human health. Here we propose using whole-genome microarrays (WGAs) to find all methylated sequences in a eukaryotic genome; the "methylome". We will test our methods with the model plant Arabidopsis thaliana, demonstrating their usefulness for the human genome. Arabidopsis is ideal for studying DNA methylation, because it has facile genetics, a small genome, and orthologs of every human DNA methyltransferase. In contrast to the lethality of mouse DNA methyltransferase mutants, Arabidopsis can tolerate mutations that virtually eliminate methylation. Our Arabidopsis thaliana arrays tile the complete genome with 25-mer oligonucleotides, allowing us to precisely measure DNA and RNA hybridization. We will use several different reagents to identify methylated DNA sequences, each coupled with high-throughput analysis using WGAs: bisulfite treatment of genomic DNA, restriction digest with methylation-sensitive enzymes, anti-methylcytosine antibodies, and proteins that bind specifically to methylated DNA. By using complementary and independent methods, we hope to detect DNA methylation with up to single nucleotide resolution. Arabidopsis DNA methyltransferase mutants have well-characterized DNA methylation defects at several endogenous loci. We will use mutants in every major Arabidopsis DNA methyltransferase to test and verify DNA methylation detection methods. RNA silencing mutants will determine how much DNA methylation is guided by small intefering RNAs (siRNAs). To further define functional non-coding elements in the genome, we correlate changes in DNA methylation with transcription and with the presence of antisense gene transcripts, and other non-coding RNAs. Once we have identified all methylated loci, we will use novel sequence libraries to classify them as transposons, other repeats, or unique sequences. This analysis is likely to reveal functional non-coding elements that are invisible to other methods. Since methylation is critical in many mammalian gene regulation phenomena, the methods developed in this proposal will clearly move the ENCODE project toward its goal of identifying functional elements in the human genome.

描述（由申请人提供）：该提案旨在通过调查全基因组 DNA 甲基化来鉴定真核基因组中的功能性非编码元件。胞嘧啶 DNA 甲基化在基因组印记和 X 染色体失活过程中控制哺乳动物的发育基因表达，并且还沉默转座子和其他重复的 DNA 序列。特定肿瘤抑制基因的高甲基化会导致癌症，表明这项工作与人类健康的相关性。在这里，我们建议使用全基因组微阵列（WGA）来查找真核基因组中的所有甲基化序列； “甲基化组”。我们将用模型植物拟南芥来测试我们的方法，证明它们对人类基因组的有用性。拟南芥是研究 DNA 甲基化的理想植物，因为它具有简单的遗传学、较小的基因组以及每种人类 DNA 甲基转移酶的直向同源物。与小鼠 DNA 甲基转移酶突变体的致命性相反，拟南芥可以耐受几乎消除甲基化的突变。我们的拟南芥阵列用 25 聚体寡核苷酸平铺完整基因组，使我们能够精确测量 DNA 和 RNA 杂交。我们将使用几种不同的试剂来鉴定甲基化 DNA 序列，每种试剂都与使用 WGA 的高通量分析相结合：基因组 DNA 的亚硫酸氢盐处理、甲基化敏感酶的限制性消化、抗甲基胞嘧啶抗体以及与甲基化 DNA 特异性结合的蛋白质。通过使用互补和独立的方法，我们希望以单核苷酸分辨率检测 DNA 甲基化。拟南芥 DNA 甲基转移酶突变体在几个内源基因座上具有明显的 DNA 甲基化缺陷。我们将使用每种主要拟南芥 DNA 甲基转移酶的突变体来测试和验证 DNA 甲基化检测方法。 RNA 沉默突变体将决定有多少 DNA 甲基化是由小干扰 RNA (siRNA) 引导的。为了进一步定义基因组中的功能性非编码元件，我们将 DNA 甲基化的变化与转录以及反义基因转录本和其他非编码 RNA 的存在联系起来。一旦我们确定了所有甲基化位点，我们将使用新的序列库将它们分类为转座子、其他重复序列或独特序列。这种分析可能会揭示其他方法看不见的功能性非编码元素。由于甲基化在许多哺乳动物基因调控现象中至关重要，因此本提案中开发的方法将明显推动 ENCODE 项目朝着识别人类基因组中功能元件的目标迈进。