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: 8081816
• 负责人: Nicolas L Young
• 金额: $ 4.3万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8081816
• 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）：我们建议采用一种新方法研究DNA损伤反应和修复机制，该方法专注于组蛋白总修饰状态中编码的早期信号，并通过我们实验室最近开发的新分析能力实现。组蛋白是构成染色体的染色质纤维的主要蛋白质组分，并且在介导DNA进入和分子机器向DNA的募集中起重要作用。个体组蛋白修饰（PTM）已被证明参与DNA损伤反应和修复等过程。这些PTM如何抑制信号的机制以及PTM的组合如何协同发挥作用仍然未知。虽然有强有力的和越来越多的证据的重要性的组合的修改，或组蛋白编码，一个彻底的研究，他们的行动已被技术限制。HeLa S3细胞培养物将用诱导几种不同DNA损伤反应和修复机制的化学物质处理。将在损伤诱导后的多个时间点取级分，提取组蛋白并与来自在富含15 N、13 C的重质培养基中生长的未处理细胞培养物的组蛋白以相等比例混合。将通过反相HPLC将组蛋白分离为单个组蛋白或序列变体。酶消化，例如GluC和AspN，将用于产生含有每个组蛋白的大部分修饰位点的相对大的肽，并且含有修饰的肽将从消化混合物中纯化。将使用pH梯度纳流弱阳离子交换-亲水相互作用色谱-电子转移解离质谱法分析含有数百至数百万个修饰形式或组蛋白代码的该样品，以鉴定和定量每个组蛋白代码。未经处理的样品将提供同位素标记的内标物用于定量。我们将进一步富集损伤位点附近的组蛋白编码，并使用上述阅读组蛋白编码的方法，通过FLAG标记染色质沉淀的途径对其进行潜在跟踪。在时间允许的情况下，我们将进行基于靶向shRNA的基因敲除，以验证并进一步研究组蛋白密码子在该途径的分子机制中的直接参与。 公共卫生相关性：DNA损伤反应和修复及其失败在癌症中很重要。DNA修复的信号是如何开始和传播的，以及进行修复的机制的募集还不清楚，这是我们研究的重点。这项工作可能会导致对癌症的更好的分子理解，更具体的诊断和潜在的癌症表观遗传疗法。