Development of novel spike-in controls for quantitative chromatin-associated protein profiling

项目摘要 染色质调节因子的改变与多种人类病理学相关。的能力 定量评估健康和患病细胞中的这些因子对于加速 靶向表观遗传调节的治疗（一个不断增长的研究领域，许多候选药物已经在临床上应用于治疗）。 审判）。然而，ChIP-Seq，最广泛使用的染色质基因组定位方法， 相关蛋白（ChAP）的检测通常受到分辨率、灵敏度和可靠性差的限制。史蒂文·赫尼科夫博士 一个小组最近开发了CUT&RUN（使用核酸酶进行靶下切割和释放），这是一种新的作图方法， 与ChIP-Seq相比，CUT&RUN使用ChAP靶向抗体， 局部拴蛋白A-微球菌核酸酶（pA-MNase）的染色质在完整的核，然后控制 MNase激活以切割附近的DNA。对随后释放的DNA片段进行测序， 使用所需细胞输入的分数（与ChIP-Seq相比）（低100倍）的靶定位谱，以及 测序深度（>10倍以下）。这种方法的效率现在可以在临床前应用， 高通量形式，如量化表观遗传疗法的全基因组效应。然而，在这方面， 要兑现这一承诺，就需要制定定量的“加标”措施。 在这个快速通道SBIR提案中，EpiCypher®与Henikoff的甲米艾哈迈德博士合作 实验室使用CUT&RUN（ChAP-CUT&RUN）开发ChAP的定量加标对照。EpiCypher 最近开发了DNA条形码化的重组设计者核小体（dNucs）的应用， 用于组蛋白翻译后修饰（PTM）ChIP研究的定量加标对照（即SNAP-ChIP®）。 然而，没有工具来规范化ChAP的映射数据，这构成了最大的部分， ChIP-Seq市场该项目的创新之处在于DNA条形码化的dNucs的工程化， 1）ChAP表位;或2）与组蛋白N-末端融合的短肽标签（SPT;例如FLAG） H3.然后，这些可用于捕获ChAP或SPT特异性抗体（两者通常用于ChAP）。 标测研究）。在目标1（第一阶段）中，我们将开发一套DNA条形码化的dNuc 用于ChAP定量分析的加标（例如CTCF [转录因子]和BRD 4 [染色质相互作用因子]）。 当我们在CUT&RUN中使用这些dNuc进行定量样品时，第一阶段将成功完成 标准化在目标2（第二阶段），我们将扩大/规模化生产ChAP-CUT&RUN加标对照品 面板，并应用这些试剂建立强大的工作流程，用于定量样品标准化基因组- 宽在目标3中，我们将开发和外部验证ChAP-CUT&RUN beta试剂盒。我们设想ChAP-CUT&RUN 将成为表观遗传学领域中最广泛使用的测定法之一（鉴于测定指标与 ChIP-Seq），有可能为有限（即珍贵）临床试验的常规分析打开新市场。 样品

PROJECT SUMMARY Alterations in chromatin regulators are associated with diverse human pathologies. The ability to quantitatively assess these factors in healthy and diseased cells is essential to accelerate the development of therapeutics targeting epigenetic regulation (a growing area of study, with many candidates already in clinical trials). However, ChIP-Seq, the most widely-used approach to map the genomic location of Chromatin Associated Proteins (ChAPs), is often limited by poor resolution, sensitivity, and reliability. Dr. Steven Henikoff’s group recently developed CUT&RUN (Cleavage Under Targets and Released Using Nuclease), a new mapping approach with vastly improved assay performance vs. ChIP-Seq. CUT&RUN uses ChAP-targeting antibodies to locally tether protein A-micrococcal nuclease (pA-MNase) to chromatin in intact nuclei, followed by controlled MNase activation to cleave nearby DNA. Sequencing of the subsequently released DNA fragments yields precise target localization profiles using fractions (vs. ChIP-Seq) of the required cellular input (100-fold less) and sequencing depth (>10-fold less). The efficiency of this method could now enable pre-clinical applications in a high-throughput format, such as quantifying the genome-wide effects of epigenetic therapeutics. However, delivering on such promise will require the development of quantitative spike-ins. In this Fast-Track SBIR proposal, EpiCypher® is partnering with Dr. Kami Ahmad of the Henikoff lab to develop quantitative spike-in controls for ChAPs using CUT&RUN (ChAP-CUT&RUN). EpiCypher has recently developed the application of DNA-barcoded recombinant designer nucleosomes (dNucs) as quantitative spike-in controls for histone post-translational modification (PTM) ChIP studies (i.e. SNAP-ChIP®). However, there are no tools to normalize mapping data for ChAPs, which make up the largest segment of the ChIP-Seq market. The innovation of this project is the engineering of DNA-barcoded dNucs that contain either: 1) a ChAP epitope; or 2) a Short Peptide Tag (SPT; e.g. FLAG) fused to the N-terminus of histone H3. These can then be used to capture ChAP- or SPT-specific antibodies (both commonly used for ChAP mapping studies) in a CUT&RUN workflow. In Aim 1 (Phase I), we will develop a set of DNA-barcoded dNuc spike-ins for quantitative analysis of ChAPs (e.g. CTCF [transcription factor] and BRD4 [chromatin interactor]). Phase I will be successfully completed when we use these dNucs in CUT&RUN for quantitative sample normalization. In Aim 2 (Phase II), we will expand / scale manufacturing of ChAP-CUT&RUN spike-in control panels and apply these reagents to establish robust workflows for quantitative sample normalization genome- wide. In Aim 3 we will develop and externally validate ChAP-CUT&RUN beta kits. We envision ChAP-CUT&RUN will become one of the most widely used assays in the epigenetics field (given the vast gain in assay metrics vs. ChIP-Seq), with the potential to open new markets for the routine analysis of limited (i.e. precious) clinical samples.