Do oxidative breaks accumulate at gene regulatory regions in disease?

最近的基因组学革命渴望更好地理解DNA序列的变化是如何导致疾病的，这将通过提供更好的诊断和治疗来改善健康结果。这可以通过开发用于早期诊断的智能测序面板来实现，并利用DNA修复中的漏洞来治疗癌症，或者使用基因编辑和替换来纠正神经疾病中的单基因障碍。虽然许多突变模式(特征)是由于DNA修复缺陷引起的，但有些仍然是未知的病因，它们如何导致疾病表型通常是未知的。因此，有必要定义产生这种突变的生物学机制，并确定它们与人类疾病的联系。到目前为止，许多突变签名的作用只在基因组的蛋白质编码部分进行了研究。然而，最近，我们发现在哺乳动物基因组的调节部分出现了氧化DNA断裂和基本位点，并描述了它们的修复机制(Ray等人，自然609：1038-1047,2022)。尽管氧化应激增加与几种神经疾病有关，但从这种疾病到疾病的机制目前尚不清楚。我们认为，氧化性DNA断裂可能积累在基因调控区域，这些区域可能表现出增加的突变负担，使人想起缺陷的氧化断裂修复，最终导致它们的功能障碍和疾病表型。在这个项目中，我们将研究由DNA修复蛋白缺陷引起的两种神经系统疾病--酪氨酸二酯酶I(Tdp1)和共济失调性毛细血管扩张突变(ATM)--基因组调控区域的DNA氧化断裂。我们将通过以下方式做到这一点：1.使用我们最近在Ray等人，2022中描述的基因组图谱方法，确定患者来源神经元中氧化DNA断裂的轮廓。2.通过遗传和化学操作，在患者来源的神经元的基因调节区建立内源性氧化断裂的来源3.使用我们最近在Ray等人，2022.4中描述的方法，探索未修复的氧化断裂对转录周期早期阶段的影响。确定患者来源细胞中基因调节区的突变负担是否增加，揭示氧化破坏如何影响基因调节区和转录反应，将增加我们对这两种神经疾病的病因学的理解，并为其他神经疾病的类似方法铺平道路，这些疾病的特征是氧化应激增加，但与DNA修复蛋白的突变没有直接联系，如阿尔茨海默病和痴呆症。

The recent revolution in genomics aspires to provide better understanding of how changes in the DNA sequence cause disease, which will improve health outcomes by providing better diagnostics and therapeutics. This could be achieved by developing smart sequencing panels for early diagnosis and exploiting vulnerabilities in DNA repair to treat cancer or correcting single gene disorders using gene editing and replacement in neurological disease. Although many patterns of mutation (signatures) arise due to defective DNA repair, some remain of unknown aetiology, and how they lead to disease phenotype is often unknown. Therefore, there is a need to define biological mechanisms generating such mutations and determine their link to human disease. So far, the role of many mutation signatures has only been studied in protein-coding parts of the genome. However, recently, we have found that oxidative DNA breaks and abasic sites arise in the regulatory parts of mammalian genomes and described a mechanism for their repair (Ray et al., Nature 609 :1038-1047, 2022). Although increased oxidative stress is associated with several neurological disorders, the mechanistic pathway from this to disease is currently unclear. We propose that oxidative DNA breaks may accumulate at gene regulatory regions and that these regions may exhibit increased mutational burden reminiscent of defective oxidative break repair, eventually leading to their malfunction and disease phenotypes. In this project, we will investigate oxidative DNA breaks at regulatory genomic regions in two neurological diseases caused by defects in the DNA repair proteins, tyrosyl DNA phosphodiesterase I (TDP1) and ataxia telangiectasis mutated (ATM). We will do so by: 1. Determining the profile of oxidative DNA breaks in patient derived neurons using genomic mapping methods that we recently described in Ray et al., 2022. 2. Establishing the source of endogenous oxidative breaks at gene regulatory regions in patient derived neurons, by genetic and chemical manipulations 3. Exploring the impact of unrepaired oxidative breaks on the early stages of the transcription cycle, using methods that we recently described in Ray et al., 2022.4. Determining whether the mutational burden is increased at gene regulatory regions in patient derived cells Revealing how oxidative breaks impact gene regulatory regions and transcriptional responses will increase our understanding of the aetiology of these two neurological diseases and pave the way of similar approaches in other neurological disease that feature increased oxidative stress but not directly linked to mutations of DNA repair proteins, such as Alzheimer's disease and dementia.