NOVEL ENZYME REAGENTS FOR EPIGENETICS STUDIES.

用于表观遗传学研究的新型酶试剂。

• 批准号: 8223609
• 负责人: Yu Zheng
• 金额: $ 45.18万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223609
• 关键词: Affinity; Amino Acids; Animal Model; Applications Grants; Applied Research; Back; Base Sequence; Basic Science; Binding; Biochemical; Biological Assay; Biology; Cell Differentiation process; Cleaved cell; Communities; Complex; Coupled; Cytosine; DNA; DNA Methylation; DNA Modification Process; DNA Restriction Enzymes; Development; Dissociation; Embryonic Development; Engineering; Enzymes; Epigenetic Process; Escherichia coli; Event; Family; Foundations; Genbank; Gene Expression Regulation; Genes; Genome; Genomics; Glucosyltransferase; Goals; Grant; Homologous Gene; Human; Human Genome; In Vitro; Laboratories; Length; Life; Location; Mammalian Cell; Maps; Measures; Methodology; Methods; Methylation; Modeling; Modification; Molecular; Molecular Structure; Mus; Mutagenesis; N-terminal; New England; Nucleotides; Organism; Pattern; Phase; Play; Process; Property; Protein Engineering; Protocols documentation; Reagent; Recombinants; Research; Research Methodology; Role; Screening procedure; Side; Site; Specificity; Staging; Structure; Surveys; System; Technology; Testing; Universities; Variant; Work; base; bisulfite; design; embryonic stem cell; empowered; endonuclease; epigenomics; fascinate; improved; interest; mammalian genome; member; mouse genome; mutant; next generation; novel; public health relevance; research study; restriction enzyme; stem cell differentiation

DESCRIPTION (provided by applicant): Epigenomics studies represent the next wave of efforts to decode the information embedded in genomes beyond the nucleotide sequences. Dynamic epigenomic changes, such as DNA methylation are key processes in cell differentiation, development and many pathological events. Current methods for mapping epigenetic modifications on DNA, such as bisulfite sequencing or using methylation-sensitive restriction enzymes, are either technically challenging or provide incomplete information. The proposed research in this grant application is based on a novel family of modification-dependent restriction endonucleases (REs), represented by MspJI, which we have discovered recently. Unlike other existing REs, these enzymes recognize modified cytosines in DNA and cleave at fixed distances away from the recognition sites. One of their unique properties is that they are capable of releasing short DNA fragments containing the methylated cytosines directly from the genomic DNA. Using ultra high- throughput sequencing platforms, one would be able to identify and map epigenetic modification in a reliable and quick manner. Furthermore, a significant portion of the modified cytosines in mammalian cells is in the form of 5-hydroxymethylcytosine and current methods cannot determine their presence. MspJI in combination with a 5- hydroxymethylcytosine modifying enzyme, such as beta-glucosyltransferase, can distinguish such changes. Thus, application of these enzymes can provide a foundation for the next-generation of methods for analyzing epigenomic modification. In the Phase I research, we plan to purify the recombinant enzymes and characterize their biochemical properties in detail in vitro. In the Phase II research, we plan to determine the molecular structure of the enzymes both in their apo-forms without DNA and complexed with a methylated DNA substrate. We will establish methodologies whereby these enzymes can be used to decode the DNA methylation patterns in human, mouse and a few other model organisms. We will also examine the dynamics of DNA methylation during mouse embryonic stem cell differentiation. The availability of these enzymes will allow us to answer scientifically pressing questions, such as, the sequence location of 5-hydroxymethycytosine in the human and mouse genomes. Another goal of the Phase II research is that, based on the structures and our previously established enzyme engineering protocols, we plan to isolate mutants that may have improved properties. We believe our proposed research lays out a few unique and exciting opportunities for epigenetics research and we expect the availability of these enzymes as products and as parts of kits to have a major impact for the broader biomedical community interested in studying epigenetic modifications. PUBLIC HEALTH RELEVANCE: Epigenetic DNA modifications in mammalian genomes, especially methylation, play crucial roles in gene regulation during cell differentiation. Commonly used methods, such as bisulfite sequencing, have many inherent drawbacks. The proposed research in this project aims at providing a set of novel enzymatic reagents for mapping the epigenetic landscape based on a family of newly discovered modification-dependent restriction endonucleases. We plan to characterize the biochemical properties of these enzymes and develop methods for applying them in epigenetics research. Coupled with high-throughput sequencing technologies, they promise a much simplified pipeline from which myriads of revolutionary methodologies and research can build on.

描述（由申请人提供）：表观基因组学研究代表了下一步的努力，以解码核苷酸序列以外的基因组中嵌入的信息。动态表观基因组学变化，例如DNA甲基化是细胞分化，发育和许多病理事件的关键过程。当前在DNA上映射表观遗传修饰的方法，例如亚硫酸盐测序或使用甲基化敏感限制酶，在技术上具有挑战性或提供不完整的信息。该赠款应用中提出的研究基于一个新型的依赖于修改的限制性核酸内切酶（RES），由MSPJI代表，我们最近发现。与其他现有RES不同，这些酶识别DNA中的修饰细胞和距识别位点的固定距离裂解。他们的独特特性之一是，它们能够直接从基因组DNA中释放含有甲基化细胞固醇的短DNA片段。使用超高通量测序平台，人们将能够以可靠，快速的方式识别和映射表观遗传修饰。此外，哺乳动物细胞中修饰的细胞固醇的很大一部分是5-羟基甲基胞嘧啶的形式，并且当前方法无法确定其存在。 MSPJI与5-羟基环胞嘧啶修饰酶（例如β-葡萄糖基转移酶）结合使用，可以区分此类变化。因此，这些酶的应用可以为分析表观基因组修饰的方法的下一代提供基础。在第一阶段的研究中，我们计划纯化重组酶，并在体外详细介绍其生化特性。在II期研究中，我们计划确定酶在其Apo形式中的分子结构，而无需DNA，并与甲基化的DNA底物复合。我们将建立方法论，这些酶可用于解码人，小鼠和其他一些模型生物中的DNA甲基化模式。我们还将检查小鼠胚胎干细胞分化过程中DNA甲基化的动力学。这些酶的可用性将使我们能够回答科学的紧迫问题，例如人类和小鼠基因组中5-羟基甲霉素的序列位置。第二阶段研究的另一个目标是，基于结构和先前建立的酶工程方案，我们计划隔离可能改善特性的突变体。我们认为，我们拟议的研究为表观遗传学研究提供了一些独特而令人兴奋的机会，我们希望这些酶作为产品和一部分套件的可用性对有兴趣研究表观遗传修饰感兴趣的更广泛的生物医学界产生重大影响。 公共卫生相关性：哺乳动物基因组中的表观遗传DNA修饰，尤其是甲基化，在细胞分化过程中在基因调节中起着至关重要的作用。常用的方法（例如硫酸盐测序）具有许多固有的缺点。该项目的拟议研究旨在提供一组新型的酶试剂，以根据新发现的依赖性依赖性限制性核酸内切核酸酶制定表观遗传景观。我们计划表征这些酶的生化特性，并开发用于将其应用于表观遗传学研究中的方法。再加上高通量测序技术，它们承诺了一条简化的管道，可以从中进行无数革命性的方法和研究。