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

本项目研究生物老化背景下DNA胞嘧啶的表观遗传修饰。胞嘧啶经过甲基化，在分子杂环的碳5上附着甲基官能团，产生5-甲基胞嘧啶（5- mc）。甲基化对染色质结构的影响是基因调控的一个重要特征。这可以确定一个基因是活跃的，可能被转录成mRNA还是被抑制。一小部分5-甲基胞嘧啶经过进一步修饰，成为氧化修饰的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 et al., 2014）。这些反应的产物在DNA中是不稳定的；相反，在碱基修复过程中，它们被切除并被未修饰的胞嘧啶核苷酸取代（Maiti和Drohat， 2011）。以这种方式进行的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包括多种癌症在内的各种年龄相关疾病有关（Haffner et al., 2011）。因此，甲基化或羟甲基化胞嘧啶核苷酸的比例可能是甲基化状态可以改变的疾病的诊断和预后特征。纳米孔测序技术提供了一种强大、经济、及时的基因信息测序方法。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测定方法。传统的短读测序方法可以检测甲基化和羟甲基化，但需要将长序列修饰成短序列，然后进行生物信息学重组。这对于短读取是准确的，但准确性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性。该项目将从细胞培养开始，以产生开发纳米孔检测所需的参考基因组文库。该分析随后可以针对已知甲基化和羟甲基化位点的验证数据集进行测试，然后与其他测序技术进行基准测试。依赖于我的纳米孔试验的成功，我将使用该试验来研究胞嘧啶表观遗传修饰的酶动力学，在衰老的纵向研究中使用来自人群的遗传数据产生数学模型。这将有助于研究表观遗传“时钟”：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，监督小组由三名巴斯大学的导师组成。阿黛尔·穆雷尔教授，生物学和生物化学，是首席导师。她将为5-hmC测序提供培训。数学科学系的Sandipan Roy博士是二级导师，他将为数学建模提供培训。生物学和生物化学博士Stefan Bagby是二级导师，他将在纳米孔测序方面提供培训。牛津纳米孔技术公司是支持该项目的工业合作伙伴。该合作由牛津纳米孔技术公司的高级研究员Adrien Leger共同组织。

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.