DNA Methylation in human muscle as a a response to exercise and vitamin C

Abstract

Epigenetics is the study of heritable changes in organisms leading to the modification of gene expression without altering the underlying DNA sequence. Such changes are vitally important in cellular identity and aid in regulating important cellular processes. Modifications to DNA and histone proteins include methylation, acetylation, phosphorylation, ubiquitination, serotonylation and lactylation. All such modifications can impact overall chromatin structure, of which DNA methylation in particular is a well-researched epigenetic modification. It involves the addition of a methyl group from S-adenosylmethionine (SAM), to the 5’carbon of a cytosine residue resulting in the formation of 5-methylcytosine (5mC).

Vitamin C is a cofactor for the TET (ten-eleven translocation) isoenzymes, which catalyse the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although vitamin C acts as a direct regulator of TET activity and DNA methylation fidelity, research to date has largely focused on using tissue culture model systems and rodents at the expense of human investigation. The process of DNA demethylation in mammals, regulates the dynamics of genome-wide DNA methylation and emerging research points to the potential involvement of vitamin C in the regulation of a plethora of biological functions.

Exercise is often considered an effective and non-invasive approach for improving health. It is now well known that exercise training can improve hypertension, hyperlipidaemia and stabilise oxidative stress through the up-regulation of antioxidant enzymes. DNA methylation is generally considered a mitotically stable modification which has the potential to change over prolonged periods of time (e.g., in the development of disease and during the process of aging). However, research supports short-term changes in DNA methylation which are influenced by environmental factors such as exercise participation.

The overall hypothesis for this body of work was that exercise alone, or in conjunction with vitamin C supplementation would result in alterations to DNA methylation at key genes in human muscle. To test this, our aims were to independently examine an existing dataset prior to conducting a human intervention study, in order to identify targets with an altered methylation profile and subsequently determine if any of these modified targets are controlled by methylation.

A mixture of approaches was used in the thesis, with a large component of bioinformatic analysis throughout, but wet-lab work using normal and methylation-deficient human cell lines (HCT116 and muscle cells) were also used to validate methylation changes in the lab. Additionally, a randomised controlled trial (RCT) was used to assess the effect of a combination of exercise and vitamin C.

In the first experimental chapter, reanalysis of a publicly available dataset (Lindholm et al., 2014, 10.4161/15592294.2014.982445) investigating DNA methylation changes as a response to exercise training was used to identify an overall gain in methylation following exercise. Some muscle genes with changes in methylation were subsequently confirmed to be regulated by methylation in a methylationdeficient cell line. Further analysis identified muscle-specific regions (MYH3, MYL1, TMOD4, LMOD4, TTN, ANKRD1, TRDN, ART1) showing gains in methylation along with other genes such as keratinassociated proteins. The genes showing gains in methylation affect different components required for building and maintaining muscle. These overall results support gains in methylation at gene bodies which could suggest an increase in transcription.

An independent RCT was then carried out. An overall gain in methylation following exercise was observed; however, this gain was larger in some cases than seen by Lindholm and colleagues. Further analysis again identified many muscle-specific regions (DES, NEB, MYOT, DAG1, KLHL41, LMOD1) showing gains in methylation, with other groups such as keratin-associated proteins also being identified. The overall results support gains in methylation at some gene bodies suggesting an increase in transcription, and at some promoters, where it would lead to a decrease in transcription.

Our independent investigation using both bioinformatic and tissue culture approaches also identified that oral high-dose vitamin C ingestion alone does not cause DNA demethylation to occur, however, in one arm of the RCT (conducted in males only), exercise combined with vitamin C identified three important changes which did occur; (1) there is an overall loss observed, albeit small, (2) there is also a reduction in the magnitude of gain observed at muscle-specific genes, indicating that vitamin C is countering the effect of exercise alone and (3) there is a small group of genes showing only a specific loss in the exercise and vitamin C group e.g., ADAMTS4.

The above findings could potentially be used to improve performance of athletes through the appropriate monitoring of progress. They could be used in distinguishing between trained and lesser trained individuals. Analysing DNA methylation changes before and after a period of training could detect the effectiveness of the exercise regime along with the individual’s response. Identifying changes in expression for specific genes could also further detect such effectiveness.

Overall, the work in this thesis confirms and extends previous observations that exercise can alter the muscle methylome, as well as demonstrating that methylation can directly control transcription levels for some genes. further, this is the first study to our knowledge to show an effect of vitamin C on the human methylome, here in the context of exercise.

Date of Award	Oct 2021
Original language	English
Sponsors	Department for the Economy & Ulster University Research Challenge Fund
Supervisor	Rachelle Irwin (Supervisor), Colum Walsh (Supervisor) & Gareth Davison (Supervisor)