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Epigenetic effects of single DNA base modifications

单DNA碱基修饰的表观遗传效应

基本信息

批准号：
213739613
负责人：
Professor Dr. Andriy Khobta
金额：
--
依托单位：
Institut für Ernährungswissenschaften
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/213739613?language=en
关键词：
Epigenetic effects single DNA base

项目摘要

Epigenetic alterations and the resulting deregulation of the affected genes have been implicated in the pathogenesis of numerous diseases of adulthood and advanced age, including cancers, neurological syndromes and immune disorders. Intriguingly, the gene loci demonstrating abnormal epigenetic patterns are often simultaneously affected by genetic mutations, suggesting that both kinds of alterations possibly originate from common DNA damage events earlier in life. However, it has not yet been possible to trace them back to a particular kind of DNA lesion or damaging agent. Using photochemically damaged reporter vectors, my team has provided evidence for a chromatin-mediated gene silencing mechanism that operates on the damaged DNA. In order to get insight into the precise nature of DNA lesions capable to initiate transcriptional silencing, we further refined the experimental system by inventing an efficient method for incorporation of single DNA base modifications of a pre-defined chemical structure into vector DNA. By analyses of numerous synthetic modifications, we found that the induction of transcriptional silencing is a peculiar feature of DNA lesions processed by the base excision DNA repair pathway (BER) and that this silencing is commonly initiated by the lesion-specific DNA glycosylases. Together, these results demonstrate that DNA damage and DNA repair are implicated in an epigenetic mechanism, functionally important for the regulation of gene expression. Further research will proceed in two directions. The first is the investigation of the molecular mechanism of transcriptional silencing initiated by the BER substrates. The aims will be to discover further effectors of the damage-induced gene silencing and to identify the critical biochemical and structural transitions of the template DNA. The second direction is examination of the capacities of DNA repair pathways for the removal of pre-existent DNA modifications with either acknowledged or alleged epigenetic functions. This topic is inspired by recent discovery of several mechanisms for active DNA demethylation, wherein coordinated action of DNA-modifying enzymatic activities and DNA repair pathways results in the oxidation of a stable epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine, which is successively converted to reparable pyrimidine species. Site-specific incorporation of these DNA modifications into custom designed vector DNA, opens possibilities for monitoring these transitions at a single nucleotide scale. The aims will be to assess the potentials of the pyrimidine oxidation products positioned in a regulatory gene element for the epigenetic regulation of transcription as well as to characterise the impact of DNA repair mechanisms and of collaterally present DNA lesions on the dynamics of these marks.

表观遗传改变和由此导致的受影响基因的放松调控与许多成年期和老年疾病的发病机制有关，包括癌症、神经综合征和免疫紊乱。有趣的是，表现出异常表观遗传模式的基因座往往同时受到基因突变的影响，这表明这两种变化可能源于生命早期常见的DNA损伤事件。然而，目前还不可能将它们追溯到特定类型的DNA损伤或损伤剂。利用光化学损伤的报告载体，我的团队提供了染色质介导的基因沉默机制的证据，该机制作用于受损的DNA。为了深入了解能够引发转录沉默的DNA损伤的确切性质，我们进一步完善了实验系统，发明了一种有效的方法，将预先定义的化学结构的单个DNA碱基修改纳入载体DNA。通过对大量人工修饰的分析，我们发现，诱导转录沉默是碱基切除DNA修复途径(BER)处理的DNA损伤的一个特有特征，这种沉默通常是由病变特异性DNA糖基酶启动的。综上所述，这些结果表明DNA损伤和DNA修复与表观遗传机制有关，该机制对基因表达的调控具有重要作用。进一步的研究将从两个方向进行。首先是对BER底物引发转录沉默的分子机制的研究。其目的将是进一步发现损伤诱导的基因沉默的影响因素，并确定模板DNA的关键生化和结构转变。第二个方向是检查DNA修复途径移除先前存在的具有公认或声称的表观遗传功能的DNA修饰的能力。这一课题的灵感来自于最近发现的几种活跃的DNA去甲基化机制，其中DNA修饰酶活性和DNA修复途径的协调作用导致稳定的表观遗传标记5-甲基胞嘧啶被氧化为5-羟甲基胞嘧啶，后者被相继转化为可修复的嘧啶物种。将这些DNA修饰结合到定制设计的载体DNA中，为在单核苷酸水平上监测这些转变打开了可能性。其目的将是评估位于调控基因元件中的嘧啶氧化产物对转录的表观遗传调控的潜力，以及表征DNA修复机制和平行存在的DNA损伤对这些标记动态的影响。