Mapping 5-Hydroxymethylcytosine at single-Base Resolution in Human ES Cells

描述（由申请人提供）：5-羟甲基胞嘧啶（5-hmC）是哺乳动物基因组DNA中新发现的碱基修饰。由于目前的测序方法无法区分5-甲基mc和5-甲基mc，因此当前的挑战是开发可靠的方法来确定5-甲基mc在哺乳动物基因组中的位置，这一问题最好通过采用我们开发的新的化学标记技术来解决。我们发现5-hmC的羟甲基可以用化学修饰的葡萄糖选择性地标记，使用¿-葡萄糖基转移酶（¿-GT）。这种糖基化提供了一种将功能基团（如生物素）安装到5-hmC上的策略。通过这种方式，我们可以亲和捕获含有修饰的5-hmC的DNA片段，并开发测序方法来确定5-hmC的精确位置。利用这种方法，我们绘制了人类胚胎干细胞中5-hmC的全基因组分布图谱。在我们早期成功的基础上，我们建议开发单碱基分辨率检测和测序方法，以揭示5-hmC在人类胚胎干细胞中的分布。我们提出了两种不同的方法，都利用我们开发的选择性化学标签策略。在一种方法中，我们将用大块基团标记5-hmC，以阻止连接和线性PCR反应，从而实现5-hmC的单碱基分辨率检测。线性PCR方法可应用于Illumina测序，对人胚胎干细胞中的5-hmC进行高通量测定。在另一种方法中，我们将开发一种外切酶酶切阻断方法，使用Illumina测序检测未消化DNA片段3'端修饰的5-hmC；我们已经证明，化学修饰的5-hmC在修饰的5-hmC的3'端阻断了外切酶III的酶切。本R21应用程序中提出的新技术不仅使我们能够在人类ES细胞中以单碱基分辨率绘制5-hmC，而且还可以应用于其他样品以系统地绘制5-hmC。

DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. Because current sequencing methods cannot differentiate 5-mC from 5-hmC, the immediate challenge is to develop robust methods to ascertain the positions of 5-hmC within the mammalian genome, a problem best addressed by adapting a new chemical labeling technology that we have developed. We show that the hydroxymethyl group of 5-hmC can be selectively labeled with chemically modified glucoses using ¿-glucosyltransferase (¿-GT). This glycosylation offers a strategy of installing functional groups such as biotin onto 5-hmC. In this way, we can affinity capture DNA fragments containing the modified 5-hmC and develop sequencing methods to determine the precise locations of 5-hmC. Using this approach we have mapped the genome-wide distribution of 5-hmC in human ES cells. Building on our early successes here we propose to develop single base-resolution detection and sequencing methods to reveal the distribution of 5-hmC in human ES cells. We propose two different approaches, both utilizing the selective chemical labeling strategy we have developed. In one approach, we will label 5-hmC with bulky groups to hinder ligation and linear PCR reactions, thus achieving single base-resolution detection of 5-hmC. The linear PCR approach can be adapted into Illumina sequencing to perform high throughput determination of 5-hmC in human ES cells. In an alternative approach, we will develop an exonuclease digestion blockage method that detects modified 5-hmC at 3' end of undigested DNA fragments using Illumina sequencing; we have already shown that the chemically modified 5-hmC blocks exonuclease III digestion at 3' end of the modified 5-hmC. The new technologies proposed in this R21 application not only enable us to map 5-hmC at single-base resolution in human ES cells, but also could be applied to other samples to map 5-hmC systemically. PUBLIC HEALTH RELEVANCE: 5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification surprisingly abundant in the genomic DNAs of embryonic stem cells. The proposed work will develop efficient chemical labeling methods to perform single base-resolution detection and sequencing of 5-hmC in human ES cells. The success of the proposed work will reveal the fundamental role(s) of 5-hmC in stem cells.