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

描述(申请人提供)：我们建议用一种新的方法来研究DNA损伤反应和修复机制，该方法专注于在组蛋白总修饰状态下编码的早期信号，并利用我们实验室最近开发的新的分析能力。组蛋白是组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DNA分子机器的募集中发挥着重要作用。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如何转导信号以及PTM的组合如何协同发挥作用的机制仍不清楚。尽管有越来越多的强有力的证据表明修饰或组蛋白密码组合的重要性，但由于技术上的限制，对其作用的彻底研究一直受到阻碍。培养的HeLa S3细胞将被化学物质处理，这些化学物质可以诱导几种不同的DNA损伤反应和修复机制。在损伤诱导后的多个时间点取部分，提取组蛋白，并与在15N，13C富含重介质中生长的未经处理的细胞培养的组蛋白等比例混合。组蛋白将通过反相高效液相色谱分离成单独的组蛋白蛋白或序列变体。酶消化，例如GluC和AspN，将被用来产生相对较大的肽，这些肽包含每个组蛋白的大部分修饰位点，并且包含修饰的肽将从消化混合物中得到纯化。这一样品包含数亿到数百万种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-亲水相互作用层析-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标用于定量。我们将进一步丰富损伤部位附近的组蛋白密码，并可能通过旗帜标记染色质沉淀的途径追踪它们，使用上面读取组蛋白密码的方法。在时间允许的情况下，我们将进行基于shRNA的靶向基因敲除，以验证并进一步研究组蛋白密码在这一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是重要的。DNA修复的信号是如何开始和传播的，以及执行修复的机械的招募还不清楚，这是我们研究的重点。这项工作可能会导致对癌症更好的分子理解，更具体的诊断，以及潜在的癌症表观遗传疗法。

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.