Ultrasensitive multiomic platform using epitope-targeted DNA methylation mapping

使用表位靶向 DNA 甲基化作图的超灵敏多组学平台

• 批准号: 10384022
• 负责人: Michael-Christopher Keogh
• 金额: $ 42.62万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384022
• 关键词: Acetylation; Adoption; Antibodies; Bar Codes; Benchmarking; Binding; Bioinformatics; Biological Assay; Biological Markers; Biological Sciences; Cell Line; Cells; Chemicals; Chromatin; Complex; DNA; DNA Integration; DNA Methylation; DNA methylation profiling; Data; Development; Diagnostic; Disease; Dose; Drug Targeting; Epigenetic Process; Epitopes; Exhibits; Freezing; Gene Expression; Genomics; Histones; K-562; Letters; Lysine; MCF7 cell; Malignant Neoplasms; Manuals; Maps; Methods; Methylation; Molecular; Monitor; New England; Noise; Nucleosomes; Pharmacotherapy; Phase; Post-Translational Protein Processing; Proteins; Protocols documentation; Regulation; Resolution; Sampling; Savings; Services; Signal Transduction; Technology; Time; Tissues; Titrations; Ubiquitination; bisulfite; bisulfite sequencing; chromatin immunoprecipitation; chromatin remodeling; clinical application; data and analysis portal; experimental study; genomic tools; high throughput screening; human embryonic stem cell; inhibitor; innovation; laboratory development; methylation pattern; multiple omics; nervous system disorder; next generation; novel; novel strategies; pre-clinical; response; targeted treatment; tool; transcription factor; user-friendly

PROJECT SUMMARY Gene expression is regulated by the complex molecular cross-talk between DNA methylation (DNAme) and other chromatin features: e.g. histone post-translational modifications (PTMs) and chromatin associated proteins (ChAPs; transcription factors and chromatin remodelers). Significantly, changes in the chromatin landscape can have a profound impact on DNAme patterning (and vice versa), and these changes are connected to development as well as a broad range of diseases (from cancer to neurological disorders). However, our understanding of how DNAme co-occurs / coordinates with additional chromatin features to control gene expression is limited by a lack of reliable genomic tools. Here, EpiCypher is partnering with New England Biolabs (NEB) to develop Targeted Enzymatic Methylation-sequencing (TEM-seqTM), an ultra-sensitive multiomic mapping technology that delivers high resolution DNAme profiles (5mC/5hmC) at epitope-defined chromatin features. EpiCypher is leading the development of ultra-sensitive genomic mapping assays that use CUT&RUN / CUT&Tag methods (under the CUTANA® platform) to generate truly quantitative data using dramatically reduced cell input and sequencing depth (>10-fold savings on each parameter vs. ChIP-seq). CUTANA assays are supported by EpiCypher’s proprietary spike-in designer nucleosome (dNuc) technology to enable technical monitoring and quantitative normalization. The key innovation of the TEM-seq project is the development of a novel multiomic workflow that marries EpiCypher’s quantitative CUTANA CUT&RUN technology with unbiased DNAme analysis using NEB’s enzymatic methyl-seq (EM-seq) approach. EM-seq utilizes the enzymatic conversion of DNAme (5mC / 5hmC) and provides a much-needed alternative to bisulfite sequencing (BS; a chemical treatment that degrades DNA and has systemic sequence biases) to generate high resolution, unbiased DNAme profiles with ~10-fold less sample input (vs. BS). In Phase I Aim 1, we will rigorously validate our TEM-seq workflow in three cell lines, benchmark results against standard CUT&RUN and EM-seq assays, and further develop EpiCypher’s spike-in controls for compatibility with TEM-seq. We will advance to Phase II when we demonstrate that TEM-seq generates highly reliable DNAme maps associated with histone PTMs and ChAPs using <50k cells and <10M reads. In Phase II Aim 2, we will expand development of spike-in control panels and develop robust protocols for a wide panel of chromatin features (using validated antibodies) and sample processing methods (fresh, frozen, and fixed), including drug treatment time-course experiments to enable clinical applications. In Phase II Aim 3, we will develop / validate a TEM-seq beta kit, and also create a data analysis portal and automated assays to accelerate commercial adoption and enable a high-throughput service offering. TEM-seq will provide a powerful new tool to expand our understanding of complex chromatin signaling, further unlocking the potential of epigenetics-targeted drugs and diagnostics.

项目摘要 基因表达受DNA甲基化（DNAme）之间复杂的分子串扰调节。 和其他染色质特征：例如组蛋白翻译后修饰（PTM）和染色质相关的 蛋白质（ChAP;转录因子和染色质重塑）。值得注意的是， 景观可以对DNA模式产生深远的影响（反之亦然），这些变化是相互关联的。 发展以及广泛的疾病（从癌症到神经系统疾病）。但我们的 理解DNAme如何与其他染色质特征共同发生/协调以控制基因 由于缺乏可靠的基因组工具，表达受到限制。在这里，EpiCypher与新英格兰生物实验室合作 (NEB)开发靶向酶促甲基化测序（TEM-seqTM），一种超灵敏的多组 在表位定义的位置提供高分辨率DNAme图谱（5 mC/5 hmC）的映射技术 染色质特征EpiCypher正在领导超灵敏基因组图谱分析的开发， CUT&RUN / CUT&Tag方法（在CUTANA®平台下），使用 显著降低细胞输入和测序深度（与ChIP-seq相比，每个参数节省>10倍）。 CUTANA检测得到EpiCypher专有的加标设计核小体（dNuc）技术的支持， 实现技术监控和量化标准化。TEM-seq项目的关键创新在于 开发一种新型的多组学工作流程，结合EpiCypher的定量CUTANA CUT&RUN 使用NEB的酶促甲基测序（EM-seq）方法进行无偏DNA分析的技术。EM-seq 利用DNAme的酶促转化（5 mC/5 hmC），并提供急需的亚硫酸氢盐替代品 测序（BS;一种降解DNA并具有系统性序列偏差的化学处理），以产生高水平的 高分辨率、无偏的DNAme图谱，样品输入量约为BS的1/10。在第一阶段目标1中，我们将严格 在三种细胞系中验证我们的TEM-seq工作流程，将结果与标准CUT&RUN和EM-seq进行基准测试 分析，并进一步开发EpiCypher的spike-in控制与TEM-seq兼容。我们将推进到 第二阶段，当我们证明TEM-seq生成与组蛋白相关的高度可靠的DNAme图谱时， 使用<50 k细胞和<10 M读数的PTM和ChAP。在第二阶段目标2中，我们将扩大 控制面板，并为广泛的染色质特征面板开发强大的协议（使用经验证的抗体） 和样品处理方法（新鲜、冷冻和固定），包括药物处理时程实验， 实现临床应用。在第二阶段目标3中，我们将开发/验证TEM-seq beta试剂盒，并创建一个 数据分析门户和自动化分析，以加速商业应用并实现高通量 服务提供。TEM-seq将提供一个强大的新工具来扩展我们对复杂染色质的理解 信号，进一步释放表观遗传学靶向药物和诊断的潜力。