Base-resolution sequencing of 6mA in eukaryotic DNA

真核 DNA 中 6mA 的碱基分辨率测序

• 批准号: 9769836
• 负责人: Eric Lieberman Greer
• 金额: $ 22.13万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769836
• 关键词: Adenine; Antibodies; Bacteria; Bacterial DNA; Base Pairing; Binding; Biological Models; Biological Process; Biology; Buffers; Chromatin Structure; Complement; Coupled; Cytosine; DNA; DNA Methylation; DNA Modification Process; DNA Sequence; Deaminase; Deamination; Deoxyadenosines; Development; Directed Molecular Evolution; Enzyme Tests; Enzymes; Epigenetic Process; Escherichia coli; Eukaryota; Event; Gene Expression; Generations; Genome; Genomic DNA; Genomics; Guanine; High Pressure Liquid Chromatography; Immunoprecipitation; Inosine; Kinetics; Location; Maps; Mass Spectrum Analysis; Measures; Methods; Methylation; Methyltransferase; Modification; Mutagenesis; Mutation; Nucleotides; Organism; Play; Process; Regulation; Research Personnel; Resolution; Role; Scientist; Site; Specificity; Techniques; Testing; Transcription Initiation Site; Uracil; Zebrafish; base; bisulfite sequencing; cost; epigenomics; experimental study; genome wide methylation; imprint; mutant; non-genetic; novel; single molecule real time sequencing; tool; transmission process; zebrafish development

N6-methyladenine (6mA) was recently identified as a new base modification in metazoan genomic DNA. Methylation on adenine was previously thought to be restricted to unicellular organisms. The identification of this mark in metazoans has raised questions about the role of 6mA in metazoan biology. Some studies suggest that 6mA may correlate with developmental regulation, active transcription start sites, or transposon expression, and potentially carry non-genetic information across generations. However, 6mA is a rare DNA modification in eukaryotes and its role in regulating biological processes is still unclear. Because of the low level of 6mA in eukaryotes, some researchers have questioned whether it plays a role in regulating biological processes or is an evolutionary holdover from bacteria. Studies of 6mA have been slowed by a lack of practical methods for accurately examining 6mA locations. Current epigenomic mapping techniques are either prohibitively expensive or do not provide sufficient resolution and accuracy to map the rare 6mA occurrences in metazoan genomes. Here we propose to develop a new technique for identifying sites of adenine methylation genome-wide at nucleotide resolution in a cost-effect manner. We will determine the utility of this technique first in bacteria and then apply it to examine 6mA distribution during development in zebrafish, where we have found that it changes dramatically. This technique will provide a new tool for scientists to define 6mA sites in metazoans to enable the study of its potential role in epigenetics, imprinting and development.

N6-甲基腺嘌呤 (6mA) 最近被鉴定为后生动物基因组中的新碱基修饰 脱氧核糖核酸。以前认为腺嘌呤的甲基化仅限于单细胞生物。这 在后生动物中鉴定出该标记引发了关于 6mA 在后生动物中作用的疑问 生物学。一些研究表明 6mA 可能与发育调节、活跃 转录起始位点或转座子表达，并可能携带非遗传信息 跨越几代人。然而，6mA是真核生物中罕见的DNA修饰及其调节作用 生物过程仍不清楚。由于真核生物中 6mA 的水平较低，一些研究人员 质疑它是否在调节生物过程中发挥作用，或者是一种进化 细菌残留。由于缺乏实用的方法，6mA 的研究进展缓慢 准确检查 6mA 位置。目前的表观基因组图谱技术要么令人望而却步 昂贵或无法提供足够的分辨率和精度来映射罕见的 6mA 事件 后生动物基因组。在这里，我们建议开发一种识别腺嘌呤位点的新技术 以成本效益的方式在核苷酸分辨率下进行全基因组甲基化。我们将确定 首先在细菌中应用该技术，然后将其应用于检查过程中的 6mA 分布 斑马鱼的发育，我们发现它发生了巨大的变化。这项技术将 为科学家提供一种新工具来定义后生动物中的 6mA 位点，以便研究其潜力 在表观遗传学、印记和发育中的作用。