Reverse ChIP: Sequence-specific extraction of DNA-bound histones for analysis of

描述(申请人提供)：该项目开发了一种自动化的蛋白质制备技术，该技术使用磁性微粒通过与组蛋白结合的染色质将天然状态的组蛋白从特定的基因组位点分离出来。提取后，目标组蛋白被纯化，用于下游的定量质谱分析或酶联免疫吸附试验。组蛋白及其翻译后修饰由于在人类疾病，特别是癌症的发生和发展中的作用，具有很高的科学和药学价值。本文提出的“反向芯片”技术将满足表观基因组学和疾病研究的明显需求，并为潜在的组蛋白脱乙酰酶(HDAC)抑制剂的候选药物筛选提供一种新的工具。目前还不存在这样的能力。我们已经使用了一个合成的转基因来展示用序列或SNP特异的引物来靶向染色质片段的方法，并已经回收了足够数量的特定修饰的组蛋白，以便进行翻译后分析。我们进一步利用了这样一个事实，即基因组染色质的核小体密度通常因转录因子结合位点而降低。结合相邻的唯一序列元件，这为我们提供了方便和高度相关的靶点选择，用于基于引物的任何特定基因座的捕获。该方法以一种简化和基本上自动化的过程为任何与疾病相关的靶区生成高分辨率组合组蛋白编码信息。获得的信息可以导致识别目标生物标记物和伴随诊断，并大大增加了目前可用的蛋白质检测、识别和量化工具。蛋白质组组蛋白修饰和潜在基因组序列的组合信息有助于在分子水平上更好地了解癌症，以及其他人类疾病，如自身免疫和神经疾病。我们为此使用的染色质捕获和质谱分析工具都是尖端的，允许在第二阶段扩大技术规模，以提供高灵敏度和高通量的分析管道。第一个用途将是识别和表征源自肿瘤和正常生物标本的特定疾病相关基因的组蛋白修饰，例如在神经母细胞瘤中扩增的MYCN基因。由于其多功能性、灵敏度和吞吐量，质谱学是一种快速增长的工具。再加上我们直接的染色质分离程序的自动化，我们预计序列特定的组蛋白提取可以迅速成为临床环境中使用的分析方法。我们将首先研究从神经母细胞瘤细胞系制备的染色质，然后与我们的科学合作者将这项技术应用于实际的肿瘤和正常的神经母细胞瘤样本。 公共卫生相关性：组蛋白翻译后修饰(PTM)是一个为改善人类健康而深入研究的领域。这些PTM模式的改变在癌症等常见疾病的病理中扮演着不同的角色，这里开发的技术将首次允许研究人员和临床医生将组蛋白密码的独特变化与其潜在的特定基因组序列相关联。这提供了在分子水平上对表观遗传信号模式及其在疾病发展和可能的治疗中的作用的全面理解。

DESCRIPTION (provided by applicant): This project develops an automated protein preparation technology that uses magnetic microparticles to isolate histones in their native state from specific genomic loci of interest via the chromatin to which they are bound. After extraction, the targeted histones are purified for downstream analysis by quantitative mass spectrometry or ELISA. Histones and their post-translational modification are of high scientific and pharmaceutical importance due to their role in the causation and development of human disease, in particular cancer. The 'reverse ChIP' technology proposed here will fill a distinct need in epigenomics and disease research, and provide a new tool for the candidate drug screening of potential histone deacetylase (HDAC) inhibitors. Currently no such ability exists. We have used a synthetic transgene to demonstrate the approach of targeting chromatin segments with sequence- or SNP-specific primers and have recovered specifically modified histones in sufficient amount to permit post-translational analysis. We further make use of the fact that the nucleosome density of genomic chromatin is generally reduced for transcription factor binding sites. In conjunction with adjacent unique sequence elements, this provides us with convenient and highly relevant choices of target sites for the primer-based capture of any specific locus. The approach generates high-resolution combinatorial histone code information for any disease-associated target region in a streamlined and largely automated process. The information gained can lead to the identification of target biomarkers and Companion Diagnostics and significantly adds to currently available tools for protein detection, identification and quantification. The combined information of proteomic histone modification and of the underlying genomic sequence contributes to a better understanding of cancer at the molecular level, as well as of other human diseases such as autoimmune and neurological disorders. The chromatin capture and mass spectrometry tools we use for this purpose are both cutting edge and allow scaling up the technology in Phase II to provide a highly sensitive and high-throughput analysis pipeline. One first use will be identifying and characterizing histone modifications originating from specific disease- associated loci from tumor and normal biospecimens, such as the MYCN locus amplified in neuroblastoma. Mass spectrometry is a rapidly growing tool due to its versatility, sensitivity and throughput. Coupled with the automation of our straightforward chromatin isolation procedure we expect that sequence-specific histone extraction can quickly become an assay that is used in a clinical environment. We will first work on chromatin prepared from neuroblastoma cell lines, before applying the technology with our scientific collaborators to actual tumor versus normal samples of neuroblastoma. PUBLIC HEALTH RELEVANCE: Histone post-translational modifications (PTMs) are an intensely investigated field for improving human health. Alterations in these PTM patterns play distinct roles in the pathology of common diseases such as cancer, and the technology developed here will allow researchers and clinicians for the first time to correlate unique changes in the histone code to their underlying specific genomic sequence. This provides a comprehensive understanding of epigenetic signaling patterns at the molecular level and their role in the development and possible treatment of disease.