Chemical Strategies for Epigenome Wide Discrimination of 5-Hydroxymethylcytosine and 5-Methylcytosine

胞嘧啶甲基化是哺乳动物基因组的一个主要修饰，通常与基因抑制和调控有关，发育过程中5-甲基胞嘧啶模式的改变有助于基因表达和细胞规范的调节。鉴于其重要性，所谓的亚硫酸盐测序方法已经开发出来，允许比较基因组DNA的5-甲基胞嘧啶/胞嘧啶分析。然而，除了5-甲基- c胞嘧啶修饰，5-羟甲基胞嘧啶（5-羟甲基- c或5-hmC）最近被确定为一种表观遗传修饰。在人类中，产生羟甲基c的酶的突变与髓系恶性肿瘤有关。证据还表明，5-羟甲基c是“干细胞性”和神经元谱系规范的表观遗传标记。然而，目前的DNA测序技术无法区分5-甲基- c和5-羟甲基- c。事实上，可以肯定的是，以前的研究将5-羟甲基c误读为5-甲基c。5-羟甲基- c的存在不仅需要重新评估现有的DNA甲基化数据，而且需要开发一种新的方法来确定5-羟甲基- c在健康和患病状态的人体组织中的相对分布和变化。该建议的基础是开发基于化学的方法，以允许全基因组分析5-甲基- c和5-羟甲基- c。

Methylation of cytosine is a major modification of the mammalian genome generally associated with gene repression and regulation, with alteration of 5-methylcytosine patterns during development contributing to the regulation of gene expression and cell specification. Given its importance methods of so-called bisulfite sequencing have been developed that allow comparative 5-methylcytosine/cytosine analysis of Genomic DNA. However in addition to 5-methyl-C another cytosine modification, 5-hydroxymethylcytosine (5-hydroxymethyl-C or 5-hmC) has recently been identified as an epigenetic modification. In humans, mutations in the enzymes that make hydroxymethyl-C are associated with myeloid malignancies. Evidence also suggests that's that 5-hydroxymethyl-C is an epigenetic marker of "stem-cellness" and neuronal lineage specification. However current DNA sequencing techniques cannot differentiate between 5-methyl-C and 5-hydroxymethyl-C. Indeed it is certain that previous studies have misread 5-hydroxymethyl-C as 5-methyl-C. The presence of 5-hydroxymethyl-C makes it necessary to not only re-evaluate existing DNA methylation data, but also to develop a new method to determine the relative distribution and changes of 5-hydroxymethyl-C in human tissues of healthy and diseased status. The basis of this proposal is to develop chemistry based approaches to allow the genome wide analysis of 5-methyl-C and 5-hydroxymethyl-C.