Quantitative mapping of dynamic epigenetic states in rare and stimulated immune cells

稀有和刺激免疫细胞动态表观遗传状态的定量图谱

• 批准号: 10481225
• 负责人: Michael-Christopher Keogh
• 金额: $ 102.27万
• 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: 2022-08-18 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10481225
• 关键词: Antibodies; Atlases; Bar Codes; Binding; Biological Assay; Cell Line; Cell Proliferation; Cells; ChIP-seq; Chromatin; Chromatin Structure; Collaborations; DNA; Development; Disease; Epigenetic Process; Failure; Gene Expression; Genes; Genome; Histones; Immune; Immune response; Immunologics; Immunology; Inflammatory; Lysine; Manuals; Maps; Methods; Modification; Monitor; Nature; Noise; Nucleosomes; Phase; Phosphorylation; Play; Population; Post Translational Modification Analysis; Post-Translational Protein Processing; Protocols documentation; Reaction; Reagent; Recovery; Regulation; Research; Role; Running; Services; Signal Transduction; Technology; Testing; Validation; Work; base; biomarker development; cell type; chromatin immunoprecipitation; cross reactivity; drug development; epigenomics; genomic platform; improved; innovation; macrophage; novel; nuclease; tool; vaccine development; validation studies

PROJECT SUMMARY Histone post-translational modifications (PTMs) play important roles in modulating chromatin structure and gene expression, and dysregulation of these marks is associated with immune and inflammatory diseases. Recent work by Dr. Steven Josefowicz and EpiCypher highlights the dynamic regulation of histone phosphorylation (i.e. phospho-PTMs) at induced genes in stimulated primary macrophages, revealing new roles for these underappreciated marks. Indeed, our studies strongly suggest that the study of stimulation-responsive chromatin dynamics in diverse primary immune cell types will unlock novel regulatory mechanisms directly related to immune and inflammatory diseases. Phospho-PTMs and other stimulation-responsive marks are transient in nature, and must be studied in primary cells (vs. proliferative cell lines). However, analysis of PTMs (phospho-PTMs and other types) in primary immune cells has been limited by existing chromatin mapping assays (i.e. chromatin immunoprecipitation [ChIP-seq]), which display poor signal-to-noise (S/N), are extremely low-throughput and expensive, and lack the sensitivity to study rare cell populations. Even CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a new immunotethering approach that vastly outperforms ChIP-seq, still lacks the sensitivity to map histone PTMs in primary cells at low inputs (≤10K cells). To meet these needs, EpiCypher is developing EpiPrime-seqTM, an ultra-sensitive genomics platform for immunology research. A key innovation of our approach is the development of a novel CUT&RUN-based protocol to profile PTMs, including stimulation-responsive PTMs, from primary immune cells. This approach includes development of a novel "direct-to-PCR" approach that drastically improves assay sensitivity to enable low primary cell inputs (≤10K cells). We will leverage EpiCypher’s designer nucleosome (dNuc) technology to develop controls carrying diverse phospho-PTMs, enabling antibody validation and in-assay technical monitoring and normalization. For Phase I feasibility, we developed a set of phosphorylated dNucs and used them to identify highly specific phospho-PTM antibodies. These tools were used to develop EpiPrime-seq, mapping phospho- PTM at induced genes following stimulation in both abundant and rare, sorted primary immune cells. In Phase II, we will extend EpiPrime-seq development for improved sensitivity and throughput. We will develop an expanded set of phosphorylated dNuc spike-ins (PhosphoStat panel) and validate phospho-PTM antibodies. We will develop robust EpiPrime-seq protocols for ≤10K primary cells, using our novel direct-to-PCR approach to map stimulation-responsive phospho- and methyl-lysine PTMs in diverse primary immune cells. Following these studies, we will prepare for commercial release by establishing lot-release strategies and performing validation studies for EpiPrime-seq kits and PhosphoStat spike-ins. Finally, we will leverage EpiCypher’s recent advances in high-throughput CUT&RUN assays to develop automated EpiPrime-seq protocols, which we will leverage to launch assay services and create novel stimulation-responsive epigenomic atlases.

项目摘要 组蛋白翻译后修饰（PTM）在调节染色质结构中起重要作用 这些标记的基因表达以及这些标记的失调与免疫和炎症性疾病有关。 史蒂文·约瑟夫维奇（Steven Josefowicz）博士和Epicypher博士的最新工作突出了组蛋白的动态调节 在受刺激的原代巨噬细胞中诱导基因的磷酸化（即磷酸化），揭示了新的作用 对于这些基本标记。实际上，我们的研究强烈表明模拟响应的研究 潜水员的染色质动力学原理类型将直接解锁新的调节机制 与免疫和炎症性疾病有关。磷酸化和其他刺激响应标记是 本质上的瞬态，必须在原代细胞中研究（与增殖物细胞系）。但是，PTMS的分析 （磷酸化和其他类型）在原发性免疫细胞中已受到现有染色质映射的限制 测定（即染色质免疫沉淀[CHIP-SEQ]），表现出较差的信号到噪声（S/N），非常极端 低通量和昂贵，并且缺乏研究稀有细胞群体的敏感性。甚至切割（乳沟） 在靶标和使用核酸酶释放下），一种新的免疫素方式，其表现非常优于chip-seq， 仍然缺乏对低输入（≤10k细胞）的原代细胞中映射组蛋白PTM的敏感性。 为了满足这些需求，Epicypher正在开发Epiprime-Seqtm，这是一个超敏感的基因组平台 免疫学研究。我们方法的关键创新是开发一种新颖的剪切和基于运行的协议 从原发性免疫细胞中介绍PTM，包括刺激反应性PTM。这种方法包括 开发一种新颖的“直接to-pcr”方法，该方法极大地提高了测定敏感性以使其能够低 原代细胞输入（≤10k细胞）。我们将利用Epicypher的设计师核小体（DNUC）技术 开发携带潜水磷酸化PTM的控制控制，实现抗体验证和类内的技术监测 和归一化。对于第一阶段的可行性，我们开发了一组磷酸化的DNUC，并用它们来识别 高度特异性的磷酸化PTM抗体。这些工具用于开发epiprime-seq，绘制磷酸化 - 在丰富和稀有的，分类的原代免疫细胞中刺激后，PTM在诱导的基因处。在阶段 ii，我们将扩展Epiprime-seq开发，以提高灵敏度和吞吐量。我们将发展一个 扩展的磷酸化DNUC SPIKE-INS（Phospostat面板）和验证磷酸化PPTM抗体。我们 将使用我们的新型直接pcr方法来开发≤10k主细胞的稳健的附膜序列方案 潜水刺激反应性的磷酸化磷酸化磷酸化磷酸化磷酸化和甲基赖氨酸PTM。遵循这些 研究，我们将通过建立批次释放策略和执行验证来准备商业释放 附着塞克套件和磷脂尖峰的研究。最后，我们将利用Epicypher最近的进步 在高通量剪切和运行测定中以开发自动epiprime-seq协议，我们将杠杆作用 启动测定服务并创建新颖的刺激反应性表观基因组图谱。