Quantification of combinatorial epigenetic modifications using defined nucleosome standards

项目总结 组蛋白尾部的翻译后修饰(组蛋白PTM)和DNA甲基化(DNAME) 核小体形成一个复杂的分子密码，调节基因转录。对这些的异常调节 染色质的修饰与大量的人类病理有关。虽然大多数工作都是在 本领域关注的是单个修饰的签名、组蛋白PTM和/或DNAME的组合 要比单一的记号更具体、更有信息量。例如，尽管健康细胞和癌细胞 两个细胞都有H3K27me3和DNAME分布在全基因组，这两个修饰的共同定位 在癌细胞中是独一无二的。然而，现有的用于测量全球染色质修饰水平的工具是 低吞吐量，显示低灵敏度，并且无法测量组合修饰(例如免疫印迹)。 克服这些限制并与多种样品类型兼容的分析方法的发展 (包括细胞样本或血浆[用于检测循环核小体，即液体活检])将使 染色质修饰的研究可广泛用于学术、临床和制药研究。 在这里，EpiCypher将开发QuantiNucTM分析，这是一个突破性的表观遗传学平台，用于量化单个 以及直接对来自细胞或血浆样本的核小体进行组合染色质修饰。这个 这一建议的创新包括a)应用设计核小体(DNucs)系统地识别 一流的检测试剂和作为定量检测标准，b)开发重组 EpiSensors用于无偏检测DNA和DNAME，以及c)开发专有的靶向样本 高通量细胞分析的处理方法。总体而言，这个平台将提供一个量化的、低成本的 利用染色质修饰分析的成本、可扩展方法(例如，组蛋白PTM和/或DNAME) 用于染色质研究、药物开发和新的生物标记物发现。在第一阶段，我们开发了一种 针对H3K4me3 H3K27ac、共富集在活性表达的PTM的量子Nuc分析 基因。我们通过建立关键的分析方法来验证这种QuantiNuc检测的特异性和性能 人血浆中H3K4me3、H3K27ac核小体的定量参数及应用 样本。在第二阶段，我们将开发新的QuantiNuc分析方法来测量其他高价值的单项和 结合染色质修饰，并进一步验证这些检测方法用于人体血浆样本 (即液体活组织检查)。此外，我们还将开发一种新的基于细胞的靶向样本处理方法 QuantiNuc分析，这将简化细胞裂解和染色质碎裂的过程，以提供高- 吞吐量大、成本低的临床研究方法。最后，我们将为QuantiNuc的商业化推出做准备 通过组装试剂盒和执行液体活组织检查和外部验证测试进行分析 基于细胞的分析，将用于开发可靠的分析方案和产品文献。市场 这些检测方法的可获得性将改变生物标志物的发现，并加速表观遗传药物的开发。

PROJECT SUMMARY Post-translational modification of histone tails (histone PTMs) and DNA methylation (DNAme) on nucleosomes form a sophisticated molecular code that regulates gene transcription. Aberrant regulation of these chromatin modifications is associated with a vast array of human pathologies. While the majority of work in the field has focused on signatures of individual modifications, combinations of histone PTMs and/or DNAme can be more specific and informative than single marks alone. For instance, although healthy cells and cancerous cells both have H3K27me3 and DNAme distributed genome-wide, the co-localization of these two modifications occurs uniquely in cancer cells. However, existing tools to measure global levels of chromatin modifications are low-throughput, display low sensitivity, and are unable to measure combinatorial modifications (e.g. immunoblot). The development of assays that overcome these limitations and are compatible with multiple sample types (including cellular samples or plasma [for detection of circulating nucleosomes, i.e. liquid biopsy]) will make the study of chromatin modifications widely accessible for academic, clinical, and pharmaceutical research. Here, EpiCypher will develop QuantiNucTM assays, a breakthrough epigenetics platform to quantify single and combinatorial chromatin modifications directly on nucleosomes from cells or plasma samples. The innovation of this proposal includes the a) application of designer nucleosomes (dNucs) to systematically identify top-performing detection reagents and to serve as quantitative assay standards, b) development of recombinant EpiSensors for unbiased detection of DNA and DNAme, and c) development of a proprietary targeted sample processing method for high-throughput cell-based assays. Overall, this platform will provide a quantitative, low- cost, and scalable approach to leverage analysis of chromatin modifications (i.e. histone PTMs and/or DNAme) for chromatin research, drug development, and novel biomarker discovery. In Phase I, we developed a QuantiNuc assay targeting combinatorial H3K4me3+H3K27ac, PTMs that are co-enriched at actively expressed genes. We validated the specificity and performance of this QuantiNuc assay by establishing key analytical parameters and applying the assay to quantify levels of H3K4me3+H3K27ac nucleosomes from human plasma samples. In Phase II, we will develop new QuantiNuc assays to measure other high-value single and combinatorial chromatin modifications and further validate these assays for use with human plasma samples (i.e. liquid biopsy). In addition, we will develop a novel targeted sample processing method for cell-based QuantiNuc assays, which will streamline the process of cell lysis and chromatin fragmentation to deliver a high- throughput, low-cost approach for clinical research. Finally, we will prepare for commercial launch of QuantiNuc assays by assembling beta-kits and performing internal and external validation testing of both liquid biopsy and cell-based assays, which will be used to develop reliable assay protocols and product literature. Market availability of these assays will transform biomarker discovery and accelerate epigenetic drug development.