Labeling and sequencing of 5hmC and 5mC in DNA

项目概要/摘要 DNA胞嘧啶甲基化（5-甲基胞嘧啶或5 mC）是主要的表观遗传机制， 人类基因表达调控这种甲基化被人泰特家族氧化 在活性去甲基化过程中，酶转化为5-羟甲基胞嘧啶（5 hmC）。5 mC是 通常是基因抑制的标志，也用于抑制基因组中的重复元件。 人类基因组中，最近的研究表明，5 hmC倾向于标记活性位点，作为基因激活 需要去除基因体、启动子和增强子中的5 mC。5 hmC的存在 表明开放的染色质基因座，而5 mC甲基化往往标志着异染色质 地区因此，5 hmC和5 mC的基因组位置提供了全基因组范围的、全面的 关于染色质活化和阻遏的信息，其是用于分类的合适标记 单个人体细胞。5 hmC和5 mC的位置也可以存储基因的信息 在冷冻或片段化的基因组样品中的活化，例如来自活检样品或细胞的DNA， 游离DNA（cfDNA），在临床诊断和预后中提供额外的优势， 各种人类疾病。 在我们以前成功发明标记和测序技术的基础上， 5 hmC和5 mC，我们最近开发了强大的程序来映射5 hmC和相关的 使用1,000个细胞进行全基因组胞嘧啶修饰。在本申请中，我们提出了新的 可以将5 hmC和5 hmC以基本分辨率映射到单细胞水平的方法。 我们计划通过研究细胞间异质性来测试和验证这些新方法， tet 2突变型急性髓性白血病干细胞与对照组。我们还将验证 新的方法来映射5 hmC和5 mC在其他系统与有限的DNA。另外我们 提出了一种新的和非常有用的方法，用于DNA中5 mC的单细胞亚硫酸氢盐测序， 预扩增有限的DNA样本，以更高的丰度兼容大多数 下游分析方法，5 mC位点在扩增过程中忠实地复制。的 拟议中的研究将为PI的团队和更广泛的组织提供迫切需要的工具。 科学界研究生物医学研究和临床生物标志物的一系列问题 使用有限的输入DNA和单细胞水平的发现。

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.