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

项目总结 染色质调节因子的改变与多种人类病理有关。有能力 在健康和患病的细胞中定量评估这些因素对于加速 以表观遗传调控为目标的治疗(这是一个不断发展的研究领域，许多候选药物已经进入临床 试验)。然而，最广泛使用的染色质基因组定位方法ChIP-Seq 结合蛋白(CHAP)通常受到分辨率、灵敏度和可靠性差的限制。史蒂文·海尼科夫博士的 Group最近开发了Cut&Run(靶标下切割并使用核酸酶释放)，一种新的图谱 与芯片序列相比，检测性能有了极大的提高。Cut&Run使用CHAP靶向抗体 在完整的核中将蛋白A微球菌核酸酶(PA-MNase)局部拴在染色质上，然后对照 激活核糖核酸酶以裂解附近的DNA。对随后释放的DNA片段进行测序得到准确的 使用所需蜂窝输入的分数(与芯片序列相比)的目标定位配置文件(减少100倍)和 测序深度(&gt；减少10倍)。这种方法的效率现在可以使临床前应用在 高通量格式，例如量化表观遗传疗法的全基因组影响。然而， 要兑现这样的承诺，将需要开发定量的价格飙升。 在这份快速通道SBIR计划中，EpiCypher®与Henikoff的Kami Ahmad博士合作 实验室将使用Cut&Run(CHAP-Cut&Run)开发针对CHAP的定量尖峰控制。EpiCypher 最近开发了DNA条形码重组设计核小体(DNucs)作为 组蛋白翻译后修饰(PTM)芯片研究的定量尖峰对照(即SNAP-CHIP®)。 然而，没有工具来归一化CHAP的映射数据，CHAP构成了 芯片序列市场。该项目的创新之处在于设计了含有DNA条形码的dNucs 1)CHAP表位；或2)融合到组蛋白N端的短肽标签(SPT；例如FLAG) H3.然后，这些抗体可用于捕获CHAP或SPT特异性抗体(这两种抗体通常用于CHAP 映射研究)在切割和运行工作流中。在目标1(第一阶段)中，我们将开发一套DNA条形码dNuc CHAP定量分析的加入物(例如，CTCF[转录因子]和BRD4[染色质相互作用物])。 当我们将这些dNucs用于定量样品的切割和运行时，第一阶段将顺利完成 正常化。在目标2(第二阶段)中，我们将扩大/规模化生产CHAP-CUT和RUN尖峰控制 并应用这些试剂来建立强大的工作流程，以实现定量样本基因组标准化- 很宽。在目标3中，我们将开发和外部验证CHAP-CUT&RUN测试版套件。我们设想的是CHAP-CUT&RUN 将成为表观遗传学领域最广泛使用的检测方法之一(考虑到检测指标与 芯片序列)，有可能为有限(即宝贵)临床的常规分析打开新的市场 样本。

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.