Hybridization of Promoter DNA Targets in Chromatin to Discover Regulatory Protein

描述（由申请人提供）：染色质免疫沉淀（ChIP）是研究核蛋白影响基因调控机制的有力工具。ChIP可用于确定活细胞的染色质中是否存在特定的蛋白质，定位基因组DNA中蛋白质相互作用的区域，并分离含有特定靶标的染色质片段。然而，由于ChIP的基础是识别特定的蛋白质或功能基团，因此它在查询特定基因组区域的全部蛋白质补体方面的能力有限。为了克服这一限制，我们建议开发新的方法来分析染色质，使用杂交探针来靶向特定的DNA序列，而不是基于抗体的识别。我们特别感兴趣的是在核过程中可能形成替代结构的基因组DNA，特别是在含有富含g序列的基因启动子区域的应用，这些基因启动子区域已知在体外形成g四重结构。这些区域是癌症研究界许多猜测和不断增长的调查的焦点。本项目期间的具体目标是：(1)利用胰岛素相关多态性区（ILPR）模型系统，利用染色质反义再杂交（CAR）和染色质义再杂交（CSR），证明在体内捕获目标染色质DNA和相关蛋白的可行性和选择性，并优化条件；(2)评估CAR和CSR在癌症细胞和正常细胞中探测与人类c-myc癌基因启动子区域相关的蛋白质的有效性。提出的CAR/CSR技术应适用于任何感兴趣的基因组区域的靶DNA和相关蛋白的分离，并将成为染色质分析工具箱的重要补充。它们将通过靶向特定的DNA序列而不是特定的抗体靶点（如蛋白质）来补充ChIP技术，从而能够发现参与基因调控的新的蛋白质相互作用。在表观遗传调控方面也有令人兴奋的发现潜力。我们建议开发新的方法来分析染色质，活细胞细胞核中的遗传物质，使用杂交探针来靶向特定的DNA序列，而不是基于抗体的免疫化学靶标识别。鉴定与特定DNA靶点（如染色质中的基因启动子区域）相关的蛋白质的完整补体将有助于更好地理解基因调控，并发现新的生物标志物和药物靶点。

DESCRIPTION (provided by applicant): Chromatin immunoprecipitation (ChIP) is a powerful tool for investigating the mechanisms through which nuclear proteins influence gene regulation. ChIP can be used to determine whether or not a particular protein is present in the in the chromatin of a living cell, to localize the region of interaction of a protein within the genomic DNA, and to isolate chromatin fragments that contain a particular target. However, since the basis of ChIP is the recognition of particular proteins or functional groups, it is limited in its ability to interrogate the full complement of proteins in a specific genomic region. In order to overcome this limitation, we propose to develop new approaches to analysis of chromatin that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition. Our particular interest is in genomic DNA that may form alternate structures during nuclear processes, specifically in this application in gene promoter regions that contain G-rich sequences known to form G-quadruplex structures in vitro. These regions are the focus of much speculation and growing investigation in the cancer research community. The specific aims for this project period are to (1) demonstrate feasibility and selectivity and optimize conditions for in vivo capture of target chromatin DNA and associated proteins using chromatin antisense rehybridization (CAR) and chromatin sense rehybridization (CSR) with the model system of the insulin- linked polymorphic region (ILPR), and (2) evaluate the effectiveness of CAR and CSR to probe the proteins associated with the human c-myc oncogene promoter region in both cancer and normal cells. The proposed CAR/CSR techniques should be adaptable to isolation of target DNA and associated proteins in any genomic region of interest, and will be an important addition to the chromatin analysis toolbox. They will complement ChIP techniques by targeting specific DNA sequences rather than specific antibody targets such as proteins, thereby enabling discovery of new protein interactions that participate in gene regulation. There is exciting potential for discovery in epigenetic regulation as well. We propose to develop new approaches to analysis of chromatin, the genetic material in the nucleus of the living cell that uses hybridization probes to target specific DNA sequences rather than antibody-based recognition of immunochemical targets. Identification of the full complement of proteins associated with a particular DNA target of interest such as a gene promoter region in chromatin will lead to a better understanding of gene regulation and to the discovery of new biomarkers and drug targets.