Quantitative mapping of combinatorial histone modifications

组合组蛋白修饰的定量作图

基本信息

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
项目类别：
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

项目摘要

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.

项目概要核小体是染色质的重复构建块，由包裹的组蛋白八聚体组成组蛋白尾部装饰有多种翻译后修饰 (PTM)，它们一起形成组合分子语言（即“组蛋白密码”）来调节基因表达和其他这种复杂景观中的缺陷与大量的人类病理有关。尤其是癌症，组蛋白翻译后修饰 (PTM) 正在迅速成为诊断/预后指标。 H3K4me3 + H3K27me3“二价”启动子是癌症中 DNA 高甲基化的常见目标，导致重要发育调节因子的表达发生显着改变。组合）PTM 可以提供新的途径来获得具有疾病特异性的新生物标志物或药物靶标，迄今为止，单一 PTM 生物标志物的主要局限性是缺乏在体内研究双重 PTM 的工具。 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阅读器域作为下一代亲和试剂来直接检测这些突破。工具利用组合 PTM 的多价相互作用增强的亲和力，这是一种关键机制该项目的一个核心创新是体内双染色质阅读器域的应用。 EpiCypher 的重组设计核小体 (dNuc) 技术可表征 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忠实地复制了 dNucs。内源性三维核小体结构，这对于准确定义合作至关重要，我们将把我们经过验证的 EpiTandem 传感器应用于 CUT&RUN，这是一种超灵敏的染色质分析方法，可生成高质量的图谱数据，且输入显着降低测序要求与 ChIP-seq 我们将为 EpiTandem 传感器开发优化的 CUT&RUN 协议，展示了它们在研究全基因组组合 PTM 方面的实用性在第一阶段，我们开发了两种。 EpiTandem 传感器并利用 dNucs（单独和组合修改）来表征和验证其双 PTM 的精细结合特异性（与单 PTM 相比高达 90 倍），然后我们应用了 EpiTandem。将传感器和 DNA 条形码 dNuc 添加到 CUT&RUN，为基因组提供强有力的概念验证在第二阶段，我们将开发五个 EpiTandem 传感器和互补的集合。 dNuc 掺入（目标 1），并使用一系列细胞类型、样品在 CUT&RUN 测定中严格验证它们处理方法和投入，包括药物治疗时程实验，以突出其价值临床研究项目（目标 2）：在目标 3 中，我们将优化五种传感器的商业规模制造。和 dNuc 面板，组装 EpiTandem beta 试剂盒，并推出双 PTM 的内部 CUT&RUN 检测服务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、生成。为市场发布做准备的基本协议和产品文献。