Quantitative mapping of combinatorial histone modifications

组合组蛋白修饰的定量作图

• 批准号: 10324501
• 负责人: JONATHAN MICHAEL BURG
• 金额: $ 102.48万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-12 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324501
• 关键词: 3-Dimensional; Affinity; Avidity; Bar Codes; Binding; Biochemical; Biological Assay; Biological Markers; Cell Line; Cells; ChIP-seq; Chromatin; Clinical Research; Collection; Complex; DNA; Data; Defect; Detection; Development; Diagnostic; Disease; Dose; Drug Targeting; Epigenetic Process; Gene Expression; Genomics; High Pressure Liquid Chromatography; Histone Code; Histones; Human Pathology; Hypermethylation; Laboratories; Language; Literature; Malignant Neoplasms; Maps; Methods; Molecular; Noise; Nucleosomes; Pharmacotherapy; Phase; Physiological Processes; Play; Post-Translational Protein Processing; Preparation; Prognostic Marker; Proteins; Protocols documentation; Reader; Reagent; Recombinants; Research Personnel; Research Project Grants; Sampling; Services; Signal Transduction; Specificity; Structure; Tail; Technology; Testing; Time; Validation; cancer cell; cell type; combinatorial; drug discovery; experimental study; genome-wide; histone modification; improved; in vivo; inhibitor/antagonist; innovation; microgenomics; next generation; novel; novel marker; novel therapeutics; promoter; response; sensor; stability testing; tool

PROJECT SUMMARY Nucleosomes are the repeating building blocks of chromatin, composed of a histone octamer wrapped with DNA. Histone tails are decorated with a variety of post-translational modifications (PTMs), which together form a combinatorial molecular language (i.e. “the histone code”) that regulates gene expression and other physiological processes. Defects in this complex landscape are associated with vast human pathologies, most notably cancer, and histone PTMs are rapidly emerging diagnostic / prognostic indicators. For instance, H3K4me3 + H3K27me3 “bivalent” promoters are frequent targets of DNA hypermethylation in cancer, resulting in drastically altered expression of important developmental regulators. Reliable quantification of dual (i.e. combinatorial) PTMs may provide novel access to new biomarkers or drug targets with disease specificity, a major limitation of single PTM biomarkers to date. However, tools to study dual PTMs in vivo are lacking. Here, EpiCypher is developing EpiTandem™ Sensors, a first-in-class technology that uses combinations of chromatin reader domains as next-generation affinity reagents to directly detect dual PTMs. These breakout tools leverage the enhanced avidity of multivalent interactions for combinatorial PTMs, a key mechanism displayed by dual chromatin reader domains in vivo. A central innovation of this project is the application of EpiCypher’s recombinant designer nucleosomes (dNuc) technology to characterize EpiTandem Sensor binding specificity against singly- and combinatorially-modified nucleosome substrates. dNucs faithfully replicate the endogenous three-dimensional nucleosome structure, which is crucial to accurately define cooperative, multivalent chromatin interactions. We will apply our validated EpiTandem Sensors to CUT&RUN, an ultra- sensitive chromatin profiling method that generates high quality mapping data with significantly lower input and sequencing requirements vs. ChIP-seq. We will develop optimized CUT&RUN protocols for EpiTandem Sensors, demonstrating their utility to interrogate combinatorial PTMs genome-wide. In Phase I, we developed two EpiTandem Sensors and utilized dNucs (singly- and combinatorially-modified) to characterize and validate their exquisite binding specificity for dual PTMs (up to 90-fold vs. single PTMs). We then applied an EpiTandem Sensor and DNA-barcoded dNuc spike-ins to CUT&RUN, providing strong proof-of-concept for genomic mapping applications. In Phase II, we will develop a collection of five EpiTandem Sensors and complementary dNuc spike-ins (Aim 1), and rigorously validate them in CUT&RUN assays using a range of cell types, sample processing methods, and inputs, including drug treatment time course experiments to highlight their value in clinical research projects (Aim 2). In Aim 3 we will optimize commercial-scale manufacturing of the five Sensors and dNuc panels, assemble EpiTandem beta kits, and launch in-house CUT&RUN assay services for dual PTM mapping studies. We will provide beta kits to leading epigenetics laboratories for external testing, generating essential protocols and product literature in preparation for market release.

项目摘要 核小体是染色质的重复构建块，由Hisstone Octamer包裹 与DNA。组蛋白的尾巴装饰有各种翻译后修饰（PTMS），它们一起装饰 形成一种调节基因表达和其他的组合分子语言（即“组蛋白代码”） 生理过程。这个复杂景观中的缺陷与广泛的人类病理有关，大多数 值得注意的是癌症，组蛋白PTM迅速出现了诊断 /预后指标。例如， H3K4ME3 + H3K27Me3“二价”启动子通常是癌症中DNA高甲基化的靶标，导致 在重要的发育调节剂的表达发生了巨大变化中。可靠的双重量化（即 组合）PTM可以为具有疾病特异性的新生物标志物或药物靶标提供新的访问权限 迄今为止，单个PTM生物标志物的主要局限性。但是，缺乏研究体内双PTM的工具。 在这里，Epicypher正在开发Epitandem™传感器，这是一种使用组合的一流技术 染色质读取器结构域是直接检测双PTM的下一代亲和力试剂。这些突破 工具利用了组合PTM的多价交互的增强性，这是一个关键机制 由双染色质读取器域在体内显示。该项目的中心创新是应用 Epicypher的重组设计师核小体（DNUC）技术以表征表位传感器结合 针对单一和组合修饰的核小体底物的特异性。忠实地复制 内源性的三维核量解结构，这对于准确定义合作至关重要， 多价染色质相互作用。我们将应用经过验证的表姐妹传感器剪切和运行，这是一种超级 敏感的染色质分析方法，该方法生成高质量映射数据的输入明显较低，并且 测序要求与Chip-Seq。我们将为epitandem传感器开发优化的剪切和运行协议， 证明了他们询问组合PTMS基因组的效用。在第一阶段，我们开发了两个 表位传感器和利用DNUC（单人和组合修饰）以表征和验证其 双PTM的精美结合特异性（与单个PTM相比，最多90倍）。然后，我们应用了一个pepitandem 传感器和DNA-barcoded DNUC Spike-Ins剪切和运行，为基因组提供了强有力的概念证明 映射应用程序。在第二阶段，我们将开发五个epitandem传感器和完整性的集合 DNUC SPIKE-INS（AIM 1），并使用一系列细胞类型，样品严格地对其进行切割和运行测定 处理方法和输入，包括药物治疗时间课程实验，以突出其价值 临床研究项目（AIM 2）。在AIM 3中，我们将优化五个传感器的商业规模制造 和DNUC面板，组装表位beta套件，并为双PTM启动内部切割和运行测定服务 映射研究。我们将为领先的表观遗传学实验室提供beta套件以进行外部测试，生成 为市场发布准备的基本协议和产品文献。