Quantitative mapping of combinatorial histone modifications

项目摘要 核小体是染色质的重复结构单元，由一个组蛋白八聚体包裹， 用DNA组蛋白尾部被各种翻译后修饰（PTM）修饰， 形成一种组合的分子语言（即“组蛋白密码”），调节基因表达和其他 生理过程。这种复杂景观中的缺陷与大量的人类病理学有关， 尤其是癌症，组蛋白PTM是迅速出现的诊断/预后指标。比如说， H3 K4 me 3 + H3 K27 me 3“二价”启动子是癌症中DNA超甲基化的常见靶点， 重要的发育调节因子的表达发生了巨大的变化。可靠的双重量化（即 组合）PTM可以提供获得新的生物标志物或具有疾病特异性的药物靶点的新途径， 迄今为止单一PTM生物标志物的主要局限性。然而，缺乏在体内研究双PTM的工具。 在这里，EpiCypher正在开发EpiTandem™传感器，这是一种一流的技术， 作为下一代亲和试剂直接检测双PTM。这些突破 工具利用多价相互作用对组合PTM的增强亲合力，这是一种关键机制， 通过双染色质阅读器结构域在体内显示。该项目的一个核心创新是应用 EpiCypher的重组设计核小体（dNuc）技术，用于表征EpiTandem传感器结合 针对单一和组合修饰的核小体底物的特异性。dNuc忠实地复制了 内源性三维核小体结构，这是至关重要的准确定义合作， 多价染色质相互作用。我们将应用我们的验证EpiTandem传感器切割&运行，一个超 一种灵敏的染色质分析方法，以显著较低的输入生成高质量的作图数据， 测序要求与ChIP-seq.我们将为EpiTandem传感器开发优化的CUT&RUN协议， 证明了它们在全基因组范围内询问组合PTM的效用。在第一阶段，我们开发了两个 EpiTandem传感器，并利用dNucs（单独和组合修饰）来表征和验证其 对双PTM的精确结合特异性（与单PTM相比高达90倍）。然后我们将EpiTandem 传感器和DNA条形码dNuc加标到CUT&RUN，为基因组学提供强有力的概念验证。 地图应用程序。在第二阶段，我们将开发一系列五个EpiTandem传感器和互补的 dNuc掺入（Aim 1），并在CUT&RUN检测中使用一系列细胞类型对其进行严格验证，样品 处理方法和投入，包括药物治疗时程实验，以突出其在 临床研究项目（目标2）。在目标3中，我们将优化五种传感器的商业规模生产 和dNuc面板，组装EpiTandem beta试剂盒，并推出针对双PTM的内部CUT&RUN检测服务 制图研究。我们将向领先的表观遗传学实验室提供beta试剂盒进行外部测试， 为市场发布做准备的基本协议和产品文献。

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.