High Throughput Chromatin Immunoprecipitation on Formalin Fixed Paraffin Embedded

描述(由申请人提供)：表观遗传机制调节基因表达潜力，表观遗传标记的变化，如DNA甲基化和组蛋白修饰，已与各种疾病有关。组织样本(研究和临床)通常通过福尔马林固定和石蜡包埋(FFPE)保存，这种方法允许在最小控制的环境中长期保存。然而，FFPE样品的制备可能会有很大的变异性，保存条件也很苛刻，这使得FFPE样品对复杂的下游分析具有挑战性。临床FFPE样本通常伴随有有价值的信息，包括组织学、疗程和患者结果，因此它们是连接基础和临床研究的极其宝贵的来源。这项第一阶段提案的目的是提供可行性研究，以使用或新开发的转座酶相关染色质免疫沉淀(TA-CHIP)来定位DNA甲基化和组蛋白修饰模式，从FFPE样本中分离出DNA和染色质。TA-CHIP使用抗体/寡核苷酸结合物将转座酶(Tn5)靶向携带特定目标标记的基因组区域(例如DNA甲基化或组蛋白修饰)。当抗体结合时，转座酶切割附近的DNA，并将附着在抗体上的寡核苷酸粘贴到DNA中。然后，与这些寡核苷酸相对应的引物可用于扩增和生成下一代文库，用于全基因组测序。由于转座酶的剪切和粘贴能力，消除了传统CHIP-SEQ过程的几个关键步骤，从而简化了协议并将损失降至最低。这项第一阶段提案中描述的工作概述了我们将进行的实验，以从FFPE样本中获得转座酶兼容的基因组DNA和染色质，确定基因组DNA和染色质的最佳标记条件，以及确定TA-CHIP是否可以在区域特定和全球范围内丰富DNA甲基化和组蛋白标记。AIM 1将检查DNA甲基化模式，从而将重点放在基因组DNA上，而AIM 2将检查组蛋白标记(H3K4me3、H3K27me3和H3K9me3)，从而集中在染色质上。了解FFPE样本中存在的表观遗传变化将使我们不仅能够将表观遗传标记与疾病关联起来，而且还可能将疾病状态分层，以帮助预测治疗反应和复发。如果成功，这些努力将转化为提取转座酶兼容DNA和染色质所需试剂的商业化，以及这些提取所需的方案以及进行TA芯片离子FFPE样品的商业化。如果成功，我们将提交一份第二阶段的提案，在该提案中，我们的目标是扩大可用于研究FFPE样本的抗体范围，并尝试将多种抗体组合混合到单个芯片反应中。此外，我们还将这项技术应用于制备和保存方案差异很大的样品，试图确定在保存过程中保留高质量DNA和染色质的关键因素。

DESCRIPTION (provided by applicant): Epigenetic mechanisms regulate gene expression potential and alterations in epigenetic marks, such as DNA methylation and histone modifications, have been associated with a variety of diseases. Tissue samples (both research and clinical) are often preserved by formalin fixation and paraffin embedment (FFPE), which allows for long term storage in minimally controlled environments. However there can be significant variability in the preparation of FFPE samples and the preservation conditions are harsh, making FFPE samples challenging for sophisticated downstream analyses. Clinical FFPE samples are often accompanied by valuable information including, histology, treatment course and patient outcome, thus they are an incredibly valuable source for linking basic and clinical research. The goal of this Phase I proposal is to provide the feasibility studies to use or newly developed transposase associate chromatin immunoprecipitation (TA-ChIP) to localize DNA methylation and histone modification patterns in DNA and chromatin isolated from FFPE samples. TA-ChIP uses antibody/oligonucleotide conjugates to target a transposase (Tn5) to genomic regions carrying a particular mark of interest (e.g. DNA methylation or histone modification). Upon antibody binding the transposase cuts the nearby DNA and pastes the oligonucleotides that are attached to the antibody into the DNA. Primers corresponding to these oligonucleotides can then be used for amplification and generation of next-generation libraries for genome wide sequencing. Due to the cut and paste abilities of the transposase several key steps of the traditional ChIP-seq procedure are eliminated thereby streamlining the protocol and a minimizing loss. The work described in this Phase I proposal outlines the experiments we will perform to obtain transposase compatible genomic DNA and chromatin from FFPE samples, determine optimal tagmentation conditions for genomic DNA and chromatin as well as determine whether TA-CHIP can enrich for DNA methylation and histone marks on a region specific and global scale. Aim 1 will examine DNA methylation patterns thus focusing on genomic DNA while aim 2 examines histone marks (H3K4me3, H3K27me3 and H3K9me3) and thus focuses on chromatin. Understanding the epigenetic alterations that are present in FFPE samples will allow us to not only correlate epigenetic marks with disease, but also potentially in stratifying disease state as to help predict treatment response and recurrence. If successful, these efforts will be translated into commercialization of the reagents necessary for extracting transposase compatible DNA and chromatin and the protocols necessary for these extractions as well as performing TA-ChIP ion FFPE samples. If successful, we will submit a phase II proposal in which we aim to expand the range of antibodies that could be used to study FFPE samples and also attempt to multiplex a combination of antibodies into a single ChIP reaction. Furthermore, we would also apply this technology to samples in which the preparation and preservation protocols varied widely in attempt to determine the key factors in retaining high quality DNA and chromatin during the preservation procedure.