Multiplex nucleosome-based profiling for the development of next-generation chromatin labeling reagents

基于多重核小体的分析，用于开发下一代染色质标记试剂

• 批准号: 10094217
• 负责人: Zu-Wen Sun
• 金额: $ 92.02万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094217
• 关键词: Acylation; Address; Antibodies; Antibody Specificity; Arginine; Bar Codes; Binding; Biological Assay; Biomedical Research; Chromatin; Chromatin Structure; Collaborations; Complement; Couples; Data; Detection; Development; Diagnostic; Epigenetic Process; Family; Generations; Genomics; Goals; Histones; K562 Cells; Label; Letters; Libraries; Licensing; Lysine; Methods; Methylation; Nucleosomes; Peptides; Performance; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Production; Reaction; Reagent; Recombinant Antibody; Recombinants; S-nitro-N-acetylpenicillamine; Sampling; Science; Signal Transduction; Site-Directed Mutagenesis; Specificity; Technology; Testing; Time; Validation; Variant; Voice; base; candidate selection; chromatin immunoprecipitation; combinatorial; cost; cross reactivity; drug development; human disease; innovation; interest; next generation; phase 2 study; screening; success; technology development

PROJECT SUMMARY Alterations in chromatin structure are associated with many human diseases and often characterized by changes in histone post-translational modifications (PTMs). Histone PTMs are commonly analyzed by chromatin immunoprecipitation (ChIP), which relies on antibodies to enrich chromatin subsets. However, the accuracy of such antibodies is an increasing concern in the biomedical field, impeding advancements in chromatin science and related drugs / diagnostics. EpiCypher® is pioneering the development of technologies that use recombinant designer nucleosomes (dNucs), with an early focus on PTM antibody specificity testing. Using EpiCypher’s disruptive quantitative ChIP platform (SNAP-ChIP®; Sample Normalization and Antibody Profiling), we found that >80% of commercially-available PTM antibodies display a striking amount of off-target binding and low binding efficiency. These results underscore substantial problems in the selection and validation of antibodies to histone PTMs, which currently utilize histone peptides for candidate selection. Ideally, we would integrate SNAP-ChIP assays early in antibody development; however, SNAP-ChIP is low-throughput, labor intensive, and thus not suitable for antibody development. In this Direct-to-Phase II proposal we will address these deficiencies with NucleoPlex™, which couples modified dNucs to Luminex® xMAP® beads for multiplexed, high-throughput antibody screening. The innovation of this proposal is the creation of dNuc-xMAP bead panels (i.e. NucleoPlex panels) that enable both on- and off- target antibody profiling in a single reaction. In Phase I equivalent proof-of-concept studies, we conjugated barcoded xMAP beads to dNucs containing histone methyl-lysine PTMs, and used this panel to interrogate the binding of >50 commercially available ChIP-grade PTM antibodies. NucleoPlex data demonstrated strong concordance with SNAP-ChIP, at fraction of the time and cost, indicating that NucleoPlex provides accurate and rapid specificity screening of PTM antibodies in a nucleosomal context. EpiCypher is poised to make a significant breakthrough with the NucleoPlex platform, redefining histone PTM antibody development, screening, and validation. A major goal of this Phase II study is to integrate NucleoPlex into a recombinant antibody development pipeline, and demonstrate how this approach increases production of highly specific antibodies. To this end, we will first validate and scale manufacturing of NucleoPlex panels for distinct histone PTM families (Aim 1), and then apply NucleoPlex toward the development of recombinant antibodies (Aim 2). Finally, we will develop combinatorially-modified nucleosomes to interrogate antibody binding specificity in the context of adjacent PTMs, which have been shown to impact antibody binding (Aim 3). The development of NucleoPlex as a low-cost multiplexed antibody screening platform will accelerate the generation of highly specific histone PTM-targeting reagents, which will have a lasting impact on the epigenetics field and save millions of dollars annually that are wasted on low-quality detection reagents.

项目概要 染色质结构的改变与许多人类疾病有关，其特征通常是 组蛋白翻译后修饰 (PTM) 的变化通常通过染色质进行分析。 免疫沉淀 (ChIP) 依靠抗体来富集染色质子集，但准确性较差。 此类抗体在生物医学领域日益受到关注，阻碍了染色质科学的进步 EpiCypher® 正在引领重组技术的开发。 设计师核小体 (dNucs)，早期专注于使用 EpiCypher 进行 PTM 抗体特异性测试。 颠覆性定量 ChIP 平台（SNAP-ChIP®；样品标准化和抗体分析），我们发现 >80% 的市售 PTM 抗体表现出惊人的脱靶结合和低结合 这些结果强调了组蛋白抗体的选择和验证中的重大问题。 PTM，目前利用组蛋白肽进行候选选择，理想情况下，我们将整合 SNAP-ChIP。 然而，SNAP-ChIP 是低通量、劳动密集型的，因此不适合在抗体开发的早期进行检测。 适合抗体开发。 在这个直接进入第二阶段的提案中，我们将通过 NucleoPlex™ 解决这些缺陷，该技术结合了 将 dNucs 修饰为 Luminex® xMAP® 珠子，用于多重、高通量抗体筛选。 该提案的重点是创建 dNuc-xMAP 珠子面板（即 NucleoPlex 面板），该面板可以实现开启和关闭 在单次反应中进行目标抗体分析，在 I 期等效概念验证研究中，我们进行了缀合。 将带条形码的 xMAP 珠子连接到含有组蛋白甲基赖氨酸 PTM 的 dNucs 上，并使用该面板来询问 > 50 种市售 ChIP 级 PTM 抗体的结合显示出强大的能力。 与 SNAP-ChIP 一致，时间和成本仅为一小部分，表明 NucleoPlex 提供了准确且可靠的结果 在核小体背景下快速特异性筛选 PTM 抗体。 EpiCypher 准备通过 NucleoPlex 平台取得重大突破，重新定义 组蛋白 PTM 抗体的开发、筛选和验证是这项 II 期研究的主要目标。 将 NucleoPlex 集成到重组抗体开发流程中，并演示该方法如何 增加高特异性抗体的产量为此，我们将首先验证和规模化生产。 针对不同组蛋白 PTM 家族的 NucleoPlex panel（目标 1），然后应用 NucleoPlex 进行开发 最后，我们将开发组合修饰的核小体来研究。 相邻 PTM 背景下的抗体结合特异性，已被证明会影响抗体结合 （目标 3） NucleoPlex 作为低成本多重抗体筛选平台的发展将加速。 高度特异性组蛋白 PTM 靶向试剂的产生，这将对 表观遗传学领域每年节省数百万美元浪费在低质量检测试剂上。

项目成果

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

瓜氨酸组蛋白的定量：开发一种改进的检测方法来可靠地定量人血浆中的核小体 H3Cit。

• 发表时间: 2020-10
• 作者: Thålin, Charlotte;Aguilera, Katherina;Hall, Nathan W;Marunde, Matthew R;Burg, Jonathan M;Rosell, Axel;Daleskog, Maud;Månsson, Maja;Hisada, Yohei;Meiners, Matthew J;Sun, Zu;Whelihan, Matthew F;Cheek, Marcus A;Howard, Sarah A;Saxena
• 通讯作者: Saxena

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

KAT8 的复合物依赖性组蛋白乙酰转移酶活性决定了其在转录和细胞稳态中的作用。

• DOI: 10.1016/j.molcel.2021.02.012
• 发表时间: 2021-04-15
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Radzisheuskaya A;Shliaha PV;Grinev VV;Shlyueva D;Damhofer H;Koche R;Gorshkov V;Kovalchuk S;Zhan Y;Rodriguez KL;Johnstone AL;Keogh MC;Hendrickson RC;Jensen ON;Helin K
• 通讯作者: Helin K