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

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.