Quantification of combinatorial epigenetic modifications using defined nucleosome standards

项目摘要 组蛋白尾的翻译后修饰（组蛋白PTM）和DNA甲基化（DNAme）在 核小体形成调节基因转录的复杂分子密码。这些异常调节 染色质修饰与大量的人类病理学有关。虽然大多数工作在 该领域已经集中于个体修饰的特征，组蛋白PTM和/或DNAme的组合可以 比单独的单个标记更具体和信息量更大。例如，虽然健康的细胞和癌细胞 细胞都有H3 K27 me 3和DNAme分布在全基因组，这两种修饰的共定位 只发生在癌细胞中。然而，测量染色质修饰的全局水平的现有工具是 低通量，显示低灵敏度，并且不能测量组合修饰（例如免疫印迹）。 开发能够克服这些限制并与多种样品类型兼容的检测方法 （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 染色质修饰的研究广泛用于学术，临床和药学研究。 在这里，EpiCypher将开发QuantiNucTM检测，这是一个突破性的表观遗传学平台， 和直接在来自细胞或血浆样品的核小体上的组合染色质修饰。的 该提议的创新包括a）设计者核小体（dNucs）的应用，以系统地鉴定 性能最好的检测试剂并用作定量测定标准，B）开发重组 EpiSensors用于无偏检测DNA和DNAme，以及c）开发专有的靶向样品 用于高通量基于细胞的测定的处理方法。总的来说，这个平台将提供一个定量的，低- 利用染色质修饰（即组蛋白PTM和/或DNAme）分析的成本和可扩展方法 用于染色质研究、药物开发和新型生物标志物发现。在第一阶段，我们开发了一个 靶向组合H3 K4 me 3 + H3 K27 ac的QuantiNuc测定，在活性表达时共富集PTM 基因.我们通过建立关键的分析方法，验证了该QuantiNuc检测的特异性和性能。 参数和应用测定来定量来自人血浆的H3 K4 me 3 + H3 K27 ac核小体的水平 样品在第二阶段，我们将开发新的QuantiNuc检测试剂盒，以测量其他高价值的单一和 组合染色质修饰并进一步验证这些测定法用于人血浆样品 (i.e.液体活组织检查）。此外，我们将开发一种新的靶向样品处理方法，用于基于细胞的 QuantiNuc检测，它将简化细胞裂解和染色质片段化的过程，以提供高水平的 高通量、低成本的临床研究方法。最后，我们将为QuantiNuc的商业发布做准备 通过组装β试剂盒并对液体活检和 基于细胞的测定，将用于开发可靠的测定方案和产品文献。市场 这些分析的可用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.