Epigenetic Clocks: Sequencing the Epigenetic DNA Modifications Involved in Biological Ageing

这个项目在生物老化的背景下研究DNA中胞嘧啶的表观遗传修饰。胞嘧啶经历甲基化，甲基官能团附着在分子杂环的碳5上，产生5-甲基胞嘧啶(5-MC)。甲基化是基因调控的一个重要特征，因为它影响染色质的结构。这可以确定一个基因是活跃的，可能被转录成信使核糖核酸或被抑制。一小部分5-MC进行进一步修饰，氧化修饰为5-羟甲基胞嘧啶(5-HMC)。5-HMC已被证明是一个稳定的表观遗传标记，对基因调控具有独特的影响；然而，5-HMC也可以作为进一步氧化反应的中间体(Bachman等人，2014)。这些反应的产物在DNA中不稳定；相反，在碱基修复过程中，它们被切除并被未经修饰的胞嘧啶核苷酸取代(Maiti和Drohat，2011)。以这种方式主动去甲基化可以激活以前沉默的基因。有证据表明胞嘧啶核苷酸的甲基化状态与各种与年龄有关的疾病有关，包括多种癌症(Haffner等人，2011年)。因此，甲基化或羟甲基化胞嘧啶核苷酸的比例可以作为甲基化状态改变的疾病的诊断和预后特征。纳米孔测序技术为基因信息测序提供了一种可靠、经济、及时的方法。因此，我研究的第一个目标将是开发一种使用固态纳米孔测序来测量甲基化状态的分析方法。传统的短读测序方法可以检测甲基化和羟甲基化，但需要将长序列修改为短序列，然后进行生物信息重组。这对于较短的读取是准确的，但准确性会随着读取长度的增加而下降。基于纳米孔的解决方案可以快速读取长序列，而不需要修改，并且始终具有一致的准确度。该项目将从细胞培养开始，以生产开发基于纳米孔的分析所需的参考基因组文库。这种分析稍后可以在甲基化和羟甲基化位点已知的验证数据集上进行测试，然后与其他测序技术进行基准比较。依赖于我的纳米孔测试的成功，我将使用这种测试来研究胞嘧啶表观遗传修饰的酶动力学，使用人口老龄化的纵向研究中的遗传数据来产生数学模型。这将有助于表观遗传“时钟”的研究：表观遗传标记，如5-HMC，可用于确定生物年龄和疾病风险。作为一个涉及生物学、生物信息学和数学的跨学科项目，监督小组由巴斯大学的三名导师组成。生物学和生物化学的阿黛尔·穆雷尔教授是首席导师。她将为5-HMC的测序提供培训。数学科学的桑迪潘·罗伊博士是一名二级导师，他将为数学建模提供培训。生物和生物化学的Stefan Bagby博士是一名二级主管，他将提供纳米孔测序方面的培训。牛津纳米孔技术公司是支持该项目的工业合作伙伴。合作是由牛津纳米孔技术公司的高级研究员Adrien Leger共同组织的。

This project studies the epigenetic modification of cytosine in DNA in the context of biological ageing. Cytosine undergoes methylation with the attachment of a methyl functional group on carbon 5 of the molecule's heterocyclic ring, producing 5-methylcytosine (5-mC). Methylation is an important feature in gene regulation for its influence on chromatin structure. This can determine whether a gene is active and may be transcribed into mRNA or is suppressed. A small proportion of 5-mC undergoes further modification, becoming 5-hydroxymethlycytosine (5-hmC) as the result of oxidative modification. 5-hmC has been shown to be a stable epigenetic mark with unique influences on gene regulation; however, 5-hmC can also act as an intermediate in further oxidative reactions (Bachman et al., 2014). The products of these reactions are not stable in DNA; instead, they are excised and replaced with unmodified cytosine nucleotides during base repair (Maiti and Drohat, 2011). Active demethylation in this manner can activate genes that were previously silent. Evidence exists to link the methylation status of cytosine nucleotides with various age-related diseases, including multiple cancers (Haffner et al., 2011). As such, the proportions of methylated or hydroxymethylated cytosine nucleotides could be a diagnostic and prognostic feature in diseases where methylation status can be shown to change. Nanopore sequencing technology offers a robust, cost effective, and timely means of sequencing genetic information. The first objective of my research will thus be to develop an assay to measure methylation status using solid-state nanopore sequencing. Traditional short-read sequencing methods can detect methylation and hydroxymethylation but require the modification of long sequences into shorter ones, followed by bioinformatic reassembly. This is accurate for short reads, but accuracy decays with read length. Nanopore-based solutions can rapidly read long sequences without the requirement for modification and have a consistent level of accuracy throughout. The project will begin with cell culturing to produce reference genomic libraries needed to develop my nanopore-based assay. This assay can later be tested against validated datasets where sites of methylation and hydroxymethylation are known, and then benchmarked against other sequencing technologies. Dependent on the success of my nanopore assay, I will use this assay to study the enzyme kinetics of cytosine epigenetic modifications, producing mathematical models using genetic data from a population in a longitudinal study of ageing. This will contribute to research into epigenetic "clocks": where epigenetic marks such as 5-hmC may be used to determine biological age and disease risk. As a cross-disciplinary project involving biology, bioinformatics, and mathematics, the supervisory team is comprised of three University of Bath supervisors. Prof. Adele Murrell, Biology and Biochemistry, is the lead supervisor. She will contribute training for the sequencing of 5-hmC. Dr. Sandipan Roy, Mathematical Sciences, is a secondary supervisor, and will contribute training for mathematical modelling. Dr. Stefan Bagby, Biology and Biochemistry, is a secondary supervisor, and will contribute training on nanopore sequencing. Oxford Nanopore Technologies is the industrial partner supporting this project. Collaboration is co-organised by Adrien Leger, a senior researcher with Oxford Nanopore Technologies.