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，表观遗传标记的改变，如DNA甲基化和组蛋白修饰，与多种疾病相关。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，这允许在最低限度控制的环境中长期储存。然而，FFPE样品的制备可能存在显著的可变性，并且保存条件苛刻，使得FFPE样品对于复杂的下游分析具有挑战性。临床FFPE样本通常伴随着有价值的信息，包括组织学，治疗过程和患者结局，因此它们是连接基础和临床研究的非常有价值的来源。该I期提案的目标是提供使用或新开发的转座酶相关染色质免疫沉淀（TA-ChIP）定位从FFPE样品中分离的DNA和染色质中的DNA甲基化和组蛋白修饰模式的可行性研究。TA-ChIP使用抗体/寡核苷酸缀合物将转座酶（Tn 5）靶向携带特定目标标记（例如DNA甲基化或组蛋白修饰）的基因组区域。在抗体结合时，转座酶切割附近的DNA并将附着于抗体的寡核苷酸粘贴到DNA中。对应于这些寡核苷酸的引物然后可用于扩增和产生用于全基因组测序的下一代文库。由于转座酶的剪切和粘贴能力，消除了传统ChIP-seq程序的几个关键步骤，从而简化了方案并最大限度地减少了损失。本I期提案中描述的工作概述了我们将进行的实验，以从FFPE样品中获得转座酶相容的基因组DNA和染色质，确定基因组DNA和染色质的最佳标签化条件，以及确定TA-CHIP是否可以在区域特异性和全球范围内富集DNA甲基化和组蛋白标记。目的1将检查DNA甲基化模式，因此专注于基因组DNA，而目的2检查组蛋白标记（H3 K4 me 3，H3 K27 me 3和H3 K9 me 3），因此专注于染色质。了解FFPE样本中存在的表观遗传改变将使我们不仅能够将表观遗传标记与疾病相关联，而且还可能在分层疾病状态中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相容性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-ChIP离子FFPE样品。如果成功，我们将提交一份II期提案，我们的目标是扩大可用于研究FFPE样本的抗体范围，并尝试将抗体组合多重化为单一ChIP反应。此外，我们还将这项技术应用于样品中的制备和保存协议有很大的不同，试图确定在保存过程中保留高质量的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.