Genome Wide Analysis of DNA Methylation

描述（由申请人提供）：本提案旨在通过测量全基因组DNA甲基化来鉴定真核生物基因组中的功能性非编码元件。在哺乳动物基因组印迹和x染色体失活过程中，胞嘧啶DNA甲基化控制着发育基因的表达，并使转座子和其他重复DNA序列沉默。特定肿瘤抑制基因的高甲基化有助于癌症，显示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（WGAs）来发现真核生物基因组中的所有甲基化序列；“methylome”。我们将用模式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甲基化的理想选择，因为它具有易遗传，小基因组和所有人类DNA甲基转移酶的同源物。与小鼠DNA甲基转移酶突变的致命性相反，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-mer寡核苷酸覆盖整个基因组，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WGAs的高通量分析相结合：亚硫酸盐处理基因组DNA，甲基化敏感酶的限制性内切酶，抗甲基胞嘧啶抗体和特异性结合甲基化DNA的蛋白质。通过使用互补和独立的方法，我们希望检测DNA甲基化高达单核苷酸的分辨率。拟南芥DNA甲基转移酶突变体在几个内源性位点具有明显的DNA甲基化缺陷。我们将使用每个主要拟南芥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、反义基因转录物和其他非编码rna的存在联系起来。一旦我们确定了所有的甲基化位点，我们将使用新的序列文库将它们分类为转座子、其他重复序列或独特序列。这种分析很可能揭示其他方法不可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使ENCODE项目朝着确定人类基因组功能元件的目标迈进。

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.