Methylation Landscape of the Human Genome

人类基因组的信息内容通过胞嘧啶残基的共价甲基化来扩展，这在每个单倍体基因组中引入了大约3×10(7)个5-甲基胞嘧啶残基。甲基化触发将受影响的序列组装成低乙酰化和浓缩状态，从而抑制转录和重组。基因组甲基化模式的异常与癌症的发生和至少两种致命的遗传疾病有关，在小鼠中，甲基化模式的中断是致命的，并与基因组稳定性异常、基因组印迹和X失活有关。因此，要充分了解人类基因组的功能和组织，就需要了解叠加的甲基化模式。然而，对基因组甲基化模式的大规模组织还知之甚少。这导致了对胞嘧啶甲基化的生物学功能的不确定性和争议。我们建议通过后基因组方法绘制人类基因组的甲基化图景，该方法涉及到应用新的、简单的和健壮的方法来选择性地提取甲基化和未甲基化的序列。未甲基化的基因组文库将通过McrBC核酸酶从大肠杆菌中选择性地去除甲基化的序列来构建，甲基化的文库将通过多个甲基化敏感的限制性内切酶降解未甲基化的序列来构建。我们将首先绘制出21号染色体内所有高度甲基化的区域和所有非甲基化区域的图谱，在验证该方法后，将把分析扩展到基因组的其余部分。当这些数据从自动高通量测序仪中出现时，它们将被在线分析，并作为注释添加到豪斯勒博士和格伦迪博士及其同事开发的人类基因组浏览器中。McrBC文库的测序将全面覆盖所有CpG岛序列，这些序列标志着大多数基因的5‘端。这些数据将对客观定义5‘外显子和起始点非常重要，这些外显子和起始点已经很难通过纯粹的计算手段来识别。我们还开发了一种简单的消减杂交方法，用于分离不同组织或发育阶段，或正常组织和癌症组织之间差异甲基化的序列。在后一种情况下，该方法为已知的可能因甲基化而沉默的肿瘤抑制基因提供了全基因组扫描；还将识别新的候选肿瘤抑制基因。该方法还将分离已知和新的印记基因。即将完成的人类基因组序列为了解基因组甲基化模式的形态及其在人类发育和疾病中的作用提供了一个独特的机会。

The information content of the human genome is expanded by the covalent methylation of cytosine residues, which introduces approximately 3 X 10(7) residues of 5-methylcytosine per haploid genome. Methylation triggers assembly of affected sequences into a hypoacetylated and condensed state that inhibits transcription and recombination. Abnormalities of genomic methylation patterns are involved in carcinogenesis and at least two fatal genetic disorders, and disruption of methylation patterns in mice is lethal and is associated with abnormalities of genome stability, genomic imprinting, and X inactivation. A full understanding of the function and organization of the human genome will therefore require an understanding of the superimposed methylation patterns. However, the large-scale organization of genomic methylation patterns is very poorly understood. This has led to uncertainty and controversy as to the biological functions of cytosine methylation. We propose to map the methylation landscape of the human genome by a post-genomic approach that involves the application of new, simple, and robust methods for the selective extraction of methylated and unmethylated sequences. Unmethylated genomic libraries will be constructed by selective removal of methylated sequences by McrBC nuclease from E. coli, and methylated libraries will be made by degradation of unmethylated sequences by multiple methylation-sensitive restriction endonucleases. We will first map all heavily methylated regions and all unmethylated regions within chromosome 21, and upon validation of the method will extend analysis to the rest of the genome. These data will be analyzed online as they emerge from automated high-throughput sequencers and added as annotation to the human genome browser developed by Drs. Haussler and Grundy and their colleagues. Sequencing of McrBC libraries will provide full coverage of all CpG island sequences, which mark the 5' ends of most genes. These data will be of great importance in the objective definition of 5' exons and start sites, which have been difficult to identify by purely computational means. We have also developed a simple subtractive hybridization method for the isolation of sequences that are differentially methylated between tissues or developmental stages, or between normal and cancerous tissues. In the latter case, the method provides a genome- wide scan for known tumor suppressor genes that might have been silenced by methylation; new candidate tumor suppressor genes will also be identified. The method will also isolate both known and novel imprinted genes. The impending completion of the sequence of the human genome presents a unique opportunity to gain understanding of the shape of genomic methylation patterns and their role in human development and disease.