Labeling and sequencing of 5-hmC 5-caC and 5-fC in genomic DNA - Resubmission 0

基因组 DNA 中 5-hmC、5-caC 和 5-fC 的标记和测序 - 重新提交 0

基本信息

批准号：
8663606
负责人：
CHUAN HE
金额：
$ 34.73万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-07 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8663606
关键词：
Address Affect Affinity Aging Back Base Excision Repairs Biological Biological Process Biological Sciences Biology Biotin Brain Cell Line Cell Maintenance Cells Chemicals Chemistry Communities Coupled Cytosine DNA Deamination Detection Development Dioxygen Dioxygenases Engineering Ensure Enzymes Epigenetic Process Exhibits Family Fertilization Future Gene Expression Gene Expression Regulation Genetic Code Genome Genomic DNA Genomic Imprinting Genomics Glucose Glucosyltransferase Human Information Distribution Iron Label Location Maintenance Malignant Neoplasms Mammalian Cell Maps Methods Modification Mus Myelopoiesis National Human Genome Research Institute Nucleic Acids Pathway interactions Play Positioning Attribute Process Proteins Regulation Request for Applications Research Resolution Role Site Staging Technology Thymine DNA Glycosylase Time Tissues Variant X Inactivation base bisulfite demethylation embryonic stem cell frontier functional group genome-wide glycosylation human disease improved mammalian genome meetings oxidation single molecule success tool

项目摘要

DESCRIPTION (provided by applicant): 5-Hydroxymethylcytosine (5-hmC) is a newly identified base modification in mammalian genomic DNA. In certain tissues or cells it can accumulate to relatively high levels. 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 invented. 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 new method, we have obtained the first genome-wide distribution map of 5-hmC in the mammalian genome. Building on our early successes, we propose to develop single-base resolution detection and sequencing methods to reveal the exact locations of 5-hmC in mammalian genomes. We propose different but complementary approaches in order to ensure that effective methods will be available to the biological community in the near future. The genome-wide information obtained can be used to help probe the functional roles of 5-hmC, for instance potential proteins and/or transcriptional factors that may recognize 5-hmC in specific sequence contents. We have also shown that 5-hmC can be further oxidized to 5-fC and 5-caC by the TET family enzymes. Significantly, when paired with a normal G, the 5-fC and 5-caC modification can be excised by the human thymine DNA glycosylase (TDG) without the need for base deamination. The base excision repair (BER) process effectively converts these base modifications back to C in an active demethylation process. We plan to develop selective 5-fC and 5-caC labeling and sequencing methods to obtain a genome-wide distribution map of these intriguing base modifications, respectively. In TDG-deficient cells, we believe the genome-wide distribution information of 5-fC/5-caC may reveal active demethylation sites in the specific cell stages. The proposed research will develop urgently needed tools for the PI's group and the broad biology community to study one of the most cutting-edge frontiers of life sciences research: the potential functional roles of these newly discovered DNA base modifications in epigenetics, development, and various human diseases.

描述（由申请人提供）：5-羟基甲基胞嘧啶（5-HMC）是哺乳动物基因组DNA的新鉴定的基础修饰。在某些组织或细胞中，它可以积累到相对较高的水平。由于当前的测序方法无法将5-MC与5-HMC区分开，因此直接挑战是开发可靠的方法来确定哺乳动物基因组中5-HMC的位置，这是通过调整我们发明的新的化学标记技术来解决这个问题的问题。我们表明，使用 - 葡萄糖基转移酶（GT），可以用化学修饰的葡萄糖（GT）选择性地标记5 -HMC的羟基甲基。这种糖基化提供了安装功能组的策略作为5-HMC的生物素。通过这种方式，我们可以亲和力捕获包含修改的5-HMC的DNA片段并开发测序方法以确定5-HMC的精确位置。使用这种新方法，我们获得了哺乳动物基因组中5-HMC的第一个全基因组分布图。在我们的早期成功的基础上，我们建议开发单基分辨率检测和测序方法，以揭示5-HMC在哺乳动物基因组中的确切位置。我们提出了不同的但互补的方法，以确保在不久的将来为生物界提供有效的方法。获得的全基因组信息可用于帮助探测5-HMC的功能作用，例如潜在的蛋白质和/或转录因子，这些因素可以识别特定序列含量中的5-HMC。我们还表明，TET家族酶可以将5-HMC进一步氧化为5-FC和5-CAC。值得注意的是，当与正常G配对时，可以通过人胸腺胺DNA糖基化酶（TDG）切除5-FC和5-CAC修饰，而无需碱基脱氨基。基础切除修复（BER）过程有效地将这些基础修饰转换回C到C，在主动的脱甲基化过程中。我们计划开发选择性的5-FC和5-CAC标记和测序方法，以分别获得这些有趣的基础修饰的全基因组分布图。在缺乏TDG的细胞中，我们认为5-FC/5-CAC的全基因组分布信息可能会在特定细胞阶段揭示活性脱甲基化位点。拟议的研究将为PI小组和广泛的生物学社区开发急需的工具，以研究生命科学研究中最尖端的前沿研究：这些新发现的DNA基础在表观遗传学，发育和各种人类疾病中的潜在功能作用。