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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要组蛋白翻译后修饰 (PTM) 在调节染色质结构中发挥重要作用和基因表达，这些标记的失调与免疫和炎症疾病有关。 Steven Josefowicz 博士和 EpiCypher 最近的工作强调了组蛋白的动态调节受刺激的初级巨噬细胞中诱导基因的磷酸化（即磷酸化翻译后修饰）揭示了新的作用事实上，对于这些未被充分认识的标记，我们的研究强烈表明刺激反应的研究。不同原代免疫细胞类型中的染色质动态将直接解锁新的调节机制与免疫和炎症疾病相关的磷酸化翻译后修饰 (Phospho-PTM) 和其他刺激反应标记。本质上是短暂的，必须在原代细胞（相对于增殖细胞系）中进行研究。但是，需要对 PTM 进行分析。原代免疫细胞中的（磷酸化 PTM 和其他类型）受到现有染色质图谱的限制检测（即染色质免疫沉淀 [ChIP-seq]）显示出较差的信噪比 (S/N)，通量低且昂贵，并且缺乏研究稀有细胞群的敏感性，甚至 CUT&RUN（裂解）。在“使用核酸酶进行目标和释放”下），这是一种新的免疫束缚方法，其性能远远优于 ChIP-seq，仍然缺乏在低输入（≤10K 细胞）的原代细胞中绘制组蛋白 PTM 的敏感性。为了满足这些需求，EpiCypher 正在开发 EpiPrime-seqTM，这是一个超灵敏的基因组学平台，用于我们方法的一个关键创新是开发一种基于 CUT&RUN 的新型方案。分析来自原代免疫细胞的 PTM，包括刺激响应性 PTM。开发了一种新颖的“直接 PCR”方法，可显着提高检测灵敏度，从而实现低原代细胞输入（≤10K 细胞）我们将利用 EpiCypher 的设计核小体 (dNuc) 技术来开发携带多种磷酸化 PTM 的对照，从而实现抗体验证和测定中技术监控为了第一阶段的可行性，我们开发了一组磷酸化 dNuc 并用它们来识别。这些工具用于开发 EpiPrime-seq，绘制磷酸化 PTM 抗体图谱。在丰富和稀有的、分选的原代免疫细胞中受到刺激后，PTM 会诱导基因。 II，我们将扩展 EpiPrime-seq 开发以提高灵敏度和通量。我们扩展了一组磷酸化 dNuc spike-ins（PhosphoStat panel）并验证了磷酸化 PTM 抗体。将使用我们新颖的直接 PCR 方法为 ≤10K 原代细胞开发强大的 EpiPrime-seq 方案遵循这些，在不同的原代免疫细胞中绘制刺激响应的磷酸和甲基赖氨酸 PTM。研究后，我们将通过建立批量发布策略并进行验证来为商业发布做好准备 EpiPrime-seq 试剂盒和 PhosphoStat spin-ins 的研究最后，我们将利用 EpiCypher 的最新进展。在高通量 CUT&RUN 检测中开发自动化 EpiPrime-seq 协议，我们将利用该协议推出检测服务并创建新颖的刺激响应表观基因组图谱。