Investigation of the Histone Code in DNA Damage Response and Repair Mechanisms

DNA 损伤反应和修复机制中组蛋白密码的研究

• 批准号: 7808185
• 负责人: Nicolas L Young
• 金额: $ 5.22万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7808185
• 关键词: Address; Biological Process; Biology; Cations; Cell Culture Techniques; Cells; Chemicals; Chromatin; Chromatin Fiber; Chromatin Remodeling Factor; Chromatography; Chromosomes; Code; DNA; DNA Damage; DNA Repair; Diagnosis; Diagnostic; Digestion; Disease; Disease Progression; Dissociation; Early treatment; Electron Transport; Enzymes; Epigenetic Process; Eukaryota; Event; Failure; Genes; HeLa S3; Health; High Pressure Liquid Chromatography; Histone Code; Histones; Individual; Investigation; Label; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Methods; Modification; Molecular; Nucleosomes; Pathway interactions; Peptides; Phase; Play; Positioning Attribute; Post-Translational Protein Processing; Precipitation; Process; Proteins; Public Health; Reading; Regulation; Resolution; Role; Sampling; Signal Transduction; Site; Specificity; Technology; Time; Variant; Work; base; cancer therapy; combinatorial; histone modification; in vivo; knock-down; novel; novel strategies; pH gradient; protein complex; public health relevance; repaired; response; small hairpin RNA

DESCRIPTION (provided by applicant): We propose to study DNA damage response and repair mechanisms with a novel approach that is focused on early signals encoded in the total modification state of the histone proteins and enabled by novel analytical capabilities recently developed in our lab. Histones are the primary protein component of chromatin fiber that make up chromosomes and play an important role in mediating access to DNA and in the recruitment of molecular machinery to DNA. Individual histone modifications (PTMs) have been shown to be involved in DNA damage response and repair and other processes. The mechanisms for how these PTMs transduce signals and how the combinations of PTMs function in concert remain unknown. Although there is strong and growing evidence of the importance of the combinations of modifications, or Histone Codes, a thorough study of their action has been inhibited by technical limitations. HeLa S3 cell cultures will be treated with chemicals that induce a couple of different DNA damage response and repair mechanisms. Fractions will be taken at multiple time points after damage induction, histones extracted and mixed in equal proportion with histones from an untreated cell culture grown in 15N, 13C rich heavy media. The histones will be separated by reverse phase HPLC into the individual histone proteins or sequence variants. Enzymatic digestion, e.g. GluC and AspN, will be used to produce relatively large peptides that contain most of the modification sites of each histone and the peptide containing the modifications will be purified from the digestion mixture. This sample which contains hundreds to millions of modified forms, or Histone Codes will be analyzed using a pH gradient nanoflow weak cation exchange- hydrophilic interaction chromatography-electron transfer dissociation mass spectrometry method to identify and quantitate each histone code. The untreated sample will provide an isotopically labeled internal standard for quantitation. We will further enrich for Histone Codes near damage sites and potentially track them through pathways with FLAG-tag chromatin precipitation, using the method above for reading the Histone Codes. With time permitted, we will perform targeted shRNA based gene knockdown to validate and further investigate the direct involvement of Histone Codes in the molecular mechanisms of this pathway. PUBLIC HEALTH RELEVANCE: DNA damage response and repair and the failure thereof are important in cancer. How the signals for DNA repair begin and propagate and the recruitment of the machinery that performs the repair are not understood and is the focus our study. This work may result in a better molecular understanding of cancer, more specific diagnostics and potentially epigenetic therapies for cancer.

描述（由申请人提供）：我们建议用一种新颖的方法研究DNA损伤反应和修复机制，该方法专注于组蛋白总修饰状态下编码的早期信号，并通过我们实验室最近开发的新型分析能力实现。组蛋白是构成染色体的染色质纤维的主要蛋白质成分，在介导DNA的获取和分子机制向DNA的募集中发挥重要作用。个体组蛋白修饰（PTMs）已被证明参与DNA损伤反应和修复等过程。这些PTMs如何传导信号以及PTMs的组合如何协同起作用的机制仍然未知。尽管有越来越多的有力证据表明修饰组合或组蛋白密码的重要性，但由于技术限制，对其作用的深入研究一直受到阻碍。HeLa S3细胞培养物将用化学物质处理，这些化学物质可诱导几种不同的DNA损伤反应和修复机制。在损伤诱导后的多个时间点提取各组蛋白，并将其与未经处理的细胞培养物中的组蛋白按等比例混合，这些细胞培养物生长在富含15N， 13C的重培养基中。组蛋白将被反相高效液相色谱分离成单个组蛋白或序列变体。酶切，如GluC和AspN，将被用来产生相对较大的肽，其中包含每个组蛋白的大部分修饰位点，并且含有修饰的肽将从消化混合物中纯化。该样品包含数亿个修饰形式，或组蛋白编码将使用pH梯度纳米流弱阳离子交换-亲水性相互作用色谱-电子转移解离质谱法进行分析，以识别和定量每个组蛋白编码。未经处理的样品将提供同位素标记的内部标准进行定量。我们将进一步丰富损伤位点附近的组蛋白编码，并可能通过flag标记染色质沉淀的途径跟踪它们，使用上述方法读取组蛋白编码。在时间允许的情况下，我们将进行基于shRNA的靶向基因敲低，以验证和进一步研究组蛋白编码直接参与该途径的分子机制。