Labeling and sequencing of 5hmC and 5mC in DNA

项目摘要/摘要 DNA胞嘧啶甲基化(5-甲基胞嘧啶或5mC)是表观遗传学的主要机制 人类基因表达调控。这种甲基化是由人类Tet家族氧化的 酶在主动去甲基化过程中转化为5-羟甲基胞嘧啶(5HmC)。而5mC是 一种通常用于基因抑制的标记，也用于抑制基因中的重复元素 人类基因组，最近的研究表明，5hmC倾向于标记活跃的基因位点，如基因激活 需要去除基因体、启动子和增强子中的5mC。5hmC的存在 显示开放的染色质基因座，而严重的5mC甲基化通常标志着异染色质 地区。因此，5hmC和5mC的基因组位置提供了全基因组、全面的 关于染色质激活和抑制的信息，这是适合分类的标志 单个人类细胞。5hmC和5mC的位置也可以存储基因的信息 在冷冻或碎裂的基因组样本中的激活，例如来自活组织检查样本或细胞的DNA- 游离DNA(CfDNA)，在临床诊断和预后方面提供了额外的优势 各种人类疾病。 建立在我们之前在发明使能技术方面的成功的基础上，标签和序列 5hmC和5mC，我们最近开发了强大的程序来映射5hmC和相关 使用1,000个细胞对全基因组进行胞嘧啶修饰。在本申请中，我们提出了新的 可以在基本分辨率下将5hmC和5hmC映射到单电池级别的方法。 我们计划通过研究细胞间的异质性来测试和验证这些新方法。 TET2突变的急性髓系白血病干细胞与对照组。我们还将验证 在其他DNA有限的系统中定位5hmC和5mC的新方法。此外，我们 提出了一种新的、实用的DNA中5mC单细胞亚硫酸氢盐测序方法 将有限的DNA样本预扩增到更高的丰度，与大多数 下游分析方法，扩增过程中5mC位点重复。这个 拟议的研究将为PI的小组和更广泛的人提供迫切需要的工具 科学界将研究生物医学研究和临床生物标记物的一系列问题 使用有限的输入DNA和在单细胞水平上的发现。

Project Summary/Abstract DNA cytosine methylation (5-methylcytosine or 5mC) is the main epigenetic mechanism in human gene expression regulation. This methylation is oxidized by the human TET family enzymes to 5-hydroxymethylcytosine (5hmC) in an active demethylation process. While 5mC is a mark for gene repression in general and is also used to suppress repetitive elements in the human genome, recent studies showed that 5hmC tends to mark active loci, as gene activation requires removal of 5mC in gene bodies, promoters, and enhancers. The presence of 5hmC indicates open chromatin loci, whereas heavy 5mC methylation often marks heterochromatin regions. Therefore, genomic locations of 5hmC and 5mC provide genome-wide, comprehensive information on chromatin activation and repression, which are suitable marks for classifying individual human cells. The locations of 5hmC and 5mC can also store information of gene activation in frozen or fragmented genomic samples, such as DNA from biopsy samples or cell- free DNA (cfDNA), providing additional advantages in clinical diagnosis and prognosis of various human diseases. Building on our previous successes in inventing enabling technologies that label and sequence 5hmC and 5mC, we have recently developed robust procedures to map 5hmC and related cytosine modifications genome-wide using 1,000 cells. In this application we propose new approaches that can map both 5hmC and 5hmC at base-resolution down to the single-cell level. We plan to test and validate these new methods by investigating cell-cell heterogeneity using Tet2-mutant acute myeloid leukemia stem cells versus controls. We will also validate the new methods to map 5hmC and 5mC in other systems with limited DNA. In addition, we propose a new and highly useful method for single-cell bisulfite sequencing of 5mC in DNA by pre-amplify the limited DNA samples to much higher abundances compatible with most downstream analysis methods, with 5mC sites replicated faithfully during the amplification. The proposed research will provide urgently needed tools for the PI's group and the broader scientific community to study a range of questions in biomedical research and clinical biomarker discoveries using limited input DNA and at the single-cell level.