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.

项目总结 组蛋白翻译后修饰(PTM)在调节染色质结构中起着重要作用 和基因表达，这些标记的失调与免疫和炎症性疾病有关。 Steven Josefowicz博士和EpiCypher博士最近的工作强调了组蛋白的动态调节 刺激原代巨噬细胞中诱导基因的磷酸化(即磷酸化PTM)，揭示了新的作用 对于这些被低估的分数。事实上，我们的研究强烈表明，对刺激反应的研究 不同初级免疫细胞类型中的染色质动力学将直接揭示新的调控机制 与免疫和炎症性疾病有关。磷酸化PTM和其他刺激反应标记是 本质上是暂时性的，必须在原代细胞(与增殖细胞系)中进行研究。然而，对PTMS的分析 初级免疫细胞中的(磷酸-PTM和其他类型)受到现有染色质图谱的限制 显示低信噪比(S/N)的分析(即染色质免疫沉淀[CHIP-SEQ])是极其困难的 产量低，价格昂贵，缺乏研究稀有细胞群体的敏感性。均匀切割并运行(劈裂 在靶标和使用核酸酶释放下)，一种新的免疫拴系方法，其性能远远超过芯片序列， 在低输入的原代细胞(≤10K细胞)中，仍然缺乏对MAP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学平台 免疫学研究。我们方法的一个关键创新是开发了一种新的基于Cut&Run的协议 分析初级免疫细胞中的PTM，包括刺激反应性PTM。这种方法包括 一种新的“直接-聚合酶链式反应”方法的开发，该方法显著提高了检测的灵敏度，使 初级单元输入(≤10K单元)。我们将利用EpiCypher的设计核小体(DNuc)技术来 开发携带不同磷酸PTM的对照，使抗体验证和检测技术监测成为可能 和正常化。为了第一阶段的可行性，我们开发了一组磷酸化的dNucs，并使用它们来识别 高度特异的磷酸化PTM抗体。这些工具被用来开发EpiPrime-seq，绘制磷酸- PTM at在刺激后在丰富和稀有的原代免疫细胞中诱导基因。同相 我们将扩展EpiPrime-seq开发以提高敏感度和吞吐量。我们将开发一种 扩展的一组磷酸化dNuc刺激物(PhosphStat面板)，并验证磷酸化PTM抗体。我们 将为≤10K原代细胞开发强大的epi-Prime-seq协议，使用我们新的直接到聚合酶链式反应的方法来 MAP在不同的初级免疫细胞中对刺激反应的磷酸和甲基赖氨酸PTM。在此之后 研究，我们将通过建立批次发布策略和执行验证来为商业发布做准备 EpiPrime-Seq试剂盒和PhosphStat尖峰蛋白的研究。最后，我们将利用EpiCypher的最新进展 在高通量切割和运行分析中，以开发自动化的EpiPrime-Seq协议，我们将利用该协议 推出化验服务并创建新的刺激响应性表观基因组图谱。