DNA Methylation Helps Muscles Remember
Exercise is an adaptive process. Short-term skeletal muscle exercise results in alterations in activation of energy producing pathways and insulin sensitivity, allowing the body to rapidly respond to an environmental challenge. If only a single exercise session is done, then these changes are largely at the protein level and dissipate about 48 hours after. However, if the exercise is repetitive, a very different chain of events occurs and muscles begin to grow. This suggests that there is indeed a molecular mechanism to muscle memory. One of the differences between immediate and chronic exercise is that chronic exercise results in a unique de novo gene expression profile, leaving the epigenome open as a prime a candidate for muscle memory. Despite promising evidence, relatively little research has been done into how DNA methylation, particularly at the omic level, contributes to the long-term effects of chronic exercise.
In order to elucidate the molecular mechanism Kanzleiter et al. created a C57BL/6J mouse model of skeletal exercise by utilizing a motorized treadmill over 4 weeks that increased in intensity in order to keep their mice challenged and fit. It was found that this routine was an excellent representation of chronic exercise as measured by metrics, including a marked decrease in body fat. The group then performed a genome wide DNA methylation analysis of the mouse’s quadriceps muscle. Their technology of choice was reduced representation bisulfite sequencing (RRBS). RRBS consists of digesting genomic DNA with the MspI restriction to create fragments with CpGs on their ends. The CpGs were then bisulfite treated, underwent library prep that includes placing adapters on CpGs and sequenced on the Illumina HiSeq2000. RRBS allows for more comprehensive coverage than that other reduced representation methylome assays, like the 450K array. Furthermore, since RRBS allows for the sequencing to be targeted to CpGs and not other regions that aren’t of interest, it provides a price and processing advantage over whole genome bisulfite sequencing (WGBS). In this case, it allowed the team to examine 55% of the genomes promoters. This approach lead to discovery of 3692 significantly differentially methylated CpGs that represented 2762 gene promoter regions.
When the exercising mice were compared to matched sedentary controls it was found that 2762 gene promoter regions showed differentially methylated CpGs. This dataset was then integrated with the differential gene expression (3020 genes), with 361 genes and 469 CpGs overlapping for differential methylation and gene expression. Furthermore, 200 gene promoters were found to have the textbook negative correlation between expression and methylation in response to exercise with the majority being hypermethylated. Functionally, most of the genes are involved in muscle growth and differentiation, with a smaller, but distinct, fraction having a role in metabolic regulation. The top candidate genes were PlexinA2, which is involved in the regulation of myogenic regulatory factors; the muscle growing Igfbp4;and Dok7, which has a role in motor neuron innervation. When the results were analyzed from a transcription factor binding perspective, it was found that differentially methylated CpGs were significantly enriched for the muscle regulatory factors MyoD and myogenin, as well as the genomic insulator CTCF.
Ultimately, this report not only provides evidence that DNA methylation is involved in muscle memory at an omic level, but also offers a suite of novel candidates for further functional validation.
Kanzleiter T, Jähnert M, Schulze G, Selbig J, Hallahan N, Schwenk RW, & Schürmann A (2015). Exercise training alters DNA-methylation patterns in genes related to muscle growth and differentiation in mice. American journal of physiology. Endocrinology and metabolism PMID: 25805191